Eukaryotic layered vector initiation systems for production of recombinant proteins

ABSTRACT

The present invention provides compositions and methods for utilizing recombinant alphavirus vectors. Also disclosed are compositions and methods for making and utilizing eukaryotic layered vector initiation systems.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application No.08/931,783, filed Sep. 16, 1997, now abandoned which application is adivision of U.S. patent application Ser. No. 08/404,796, filed Mar. 15,1995; now U.S. Pat. No. 6,015,686 and is a continuation-in-part of U.S.patent application Ser. No. 08/376,184, filed Jan. 20, 1995, nowabandoned; which application is a continuation-in-part of U.S. patentapplication Ser. No. 08/348,472, filed Nov. 30, 1994, now abandoned;which application is a continuation-in-part of U.S. patent applicationSer. No. 08/198,450, filed Feb. 18, 1994, now abandoned; whichapplication is a continuation-in-part of U.S. patent application Ser.No. 08/122,791, filed Sep. 15, 1993, now abandoned, all of which areincorporated by reference in their entirety.

TECHNICAL FIELD

The present invention relates generally to use of recombinant viruses asvectors, and more specifically, to recombinant alphaviruses which arecapable of expressing a heterologous sequence in target cells.

BACKGROUND OF THE INVENTION

Alphaviruses comprise a set of serologically related arthropod-borneviruses of the Togavirus family. Briefly, alphaviruses are distributedworldwide, and persist in nature through a mosquito to vertebrate cycle.Birds, rodents, horses, primates, and humans are among the definedalphavirus vertebrate reservoir/hosts.

Twenty-six known viruses and virus subtypes have been classified withinthe alphavirus genus utilizing the hemagglutination inhibition (HI)assay. Briefly, the HI test segregates the 26 alphaviruses into threemajor complexes: the Venezuelan encephalitis (VE) complex, the SemlikiForest (SF) complex, and the western encephalitis (WE) complex. Inaddition, four additional viruses, eastern encephalitis (EE), BarmahForest, Middleburg, and Ndumu, receive individual classification basedon the HI serological assay.

Members of the alphavirus genus are also classified based on theirrelative clinical features in humans: alphaviruses associated primarilywith encephalitis, and alphaviruses associated primarily with fever,rash, and polyarthritis. Included in the former group are the VE and WEcomplexes, and EE. In general, infection with this group can result inpermanent sequelae, including behavior changes and learningdisabilities, or death. In the latter group is the SF complex, comprisedof the individual alphaviruses Chikungunya, O'nyong-nyong, Sindbis, RossRiver, and Mayaro. With respect to this group, although seriousepidemics have been reported, infection is in general self-limiting,without permanent sequelae.

Sindbis virus is the prototype member of the alphavirus genus of theTogavirus family. Although not usually apparent, clinical manifestationsof Sindbis virus infection may include fever, arthritis, and rash.Sindbis virus is distributed over Europe, Africa, Asia, and Australia,with the best epidemiological data coming from South Africa, where 20%of the population is seropositive. (For a review, see Peters andDalrymple, Fields Virology (2nd ed), Fields et al. (eds.), B.N. RavenPress, New York, N.Y., chapter 26, pp. 713-762). Infectious Sindbisvirus has been isolated from human serum only during an outbreak inUganda and in a single case from Central Africa.

The morphology and morphogenesis of the alphavirus genus is generallyquite uniform. In particular, the enveloped 60-65 nm particles infectmost vertebrate cells, where productive infection is cytopathic. On theother hand, infection of invertebrate cells, for example, those derivedfrom mosquitoes, does not result in any overt cytopathology. Typically,alphaviruses are propagated in BHK-21 or vero cells, where growth israpid, reaching a maximum yield within 24 hours of infection. Fieldstrains are usually isolated on primary avian embryo, for examplechicken fibroblast cultures (CEF).

The genomic RNA (49S RNA) of alphaviruses is unsegmented, of positivepolarity, approximately 11-12 kb in length, and contains a 5′ cap and a3′ polyadenylate tail. Infectious enveloped virus is produced byassembly of the viral nucleocapsid proteins onto genomic RNA in thecytoplasm, and budding through the cell membrane embedded withviral-encoded glycoproteins. Entry of virus into cells appears to occurby endocytosis through caltherin-coated pits, fusion of the viralmembrane with the endosome, release of the nucleocapsid and uncoating ofthe viral genome. During viral replication, the genomic 49S RNA servesas template for synthesis of a complementary negative strand. Thenegative strand in turn serves as template for full-length genomic RNAand for an internally initiated positive-strand 26S subgenomic RNA. Thenonstructural proteins are translated from the genomic RNA. Alphaviralstructural proteins are translated from the subgenomic 26S RNA. Allviral genes are expressed as polyproteins and processed into individualproteins by proteolytic cleavage post-translation.

The use of recombinant virus vectors (in particular, alphavirus vectors)to treat individuals requires that they be able to be transported andstored for long periods at a desired temperature, such that infectivityand viability of the recombinant virus is retained. Current methods forstoring recombinant viruses generally involve storage as liquids and atlow temperatures. Such methods present problems in Third Worldcountries, which typically do not have adequate refrigerationcapabilities. For example, each year in Africa, millions of children diefrom infectious diseases such as measles. Vaccines necessary for theprevention of these diseases cannot be distributed to the majority ofthese countries because refrigeration is not readily accessible.

In addition to storage as liquids and at low temperatures, present viralformulations often contain media components that are not desirable forinjection into patients. Consequently, there is a need in the art for amethod of preserving purified recombinant viral vector (and inparticular, alphavirus vectors) in a lyophilized form at elevatedtemperatures, and for this form to be suitable for injection intopatients.

The present invention discloses recombinant alphavirus vectors which aresuitable for use in a variety of applications, including for example,gene therapy, and further provides other related advantages.

SUMMARY OF THE INVENTION

Briefly sated, the present invention provides alphavirus vectorconstructs and alphavirus particles, as well as methods of making andutilizing the same. Within one aspect of the present invention,alphavirus vector constructs are provided comprising a 5′ promoter whichis capable of initiating the synthesis of viral RNA in vitro from cDNA,a 5′ sequence which is capable of initiating transcription of analphavirus, a nucleotide sequence encoding alphavirus non-structuralproteins, a viral junction region which has been inactivated such thatthe viral transcription of the subgenomic fragment is prevented, and analphavirus RNA polymerase recognition sequence. Within other aspects ofthe present invention, the viral junction region has been modified suchthat viral transcription of the subgenomic fragment is reduced.

Within yet other aspects of the present invention, alphavirus vectorconstructs are provided comprising a 5′ promoter which is capable ofinitiating the synthesis of viral RNA in vitro from cDNA, a 5′ sequencewhich is capable of initiating transcription of an alphavirus, anucleotide sequence encoding alphavirus non-structural proteins, a firstviral junction region which has been inactivated such that viraltranscription of the subgenomic fragment is prevented, a second viraljunction region which is active, or which has been modified such thatviral transcription of the subgenomic fragment is reduced, and analphavirus RNA polymerase recognition sequence.

Within still other aspects of the present invention, alphavirus cDNAvector constructs are provided, comprising a 5′ promoter which iscapable of initiating the synthesis of viral RNA from cDNA, followed bya 5′ sequence which is capable of initiating transcription of analphavirus, a nucleotide sequence encoding alphavirus non--structuralproteins, a viral junction region which has been inactivated such thatviral transcription of the subgenomic fragment is prevented, analphavirus RNA polymerase recognition sequence, and a 3′ sequence whichcontrols transcription termination.

Within another aspect of the present invention, alphavirus cDNA vectorconstructs are provided, comprising a 5′ promoter which is capable ofinitiating the synthesis of viral RNA from cDNA, followed by a 5′sequence which is capable of initiating transcription of an alphavirus,a nucleotide sequence encoding alphavirus non-structural proteins, aviral junction region which is active, or which has been modified suchthat viral transcription of the subgenomic fragment is reduced, analphavirus RNA polymerase recognition sequence, and a 3′ sequence whichcontrols transcription termination.

Within another aspect of the present invention, alphavirus cDNA vectorconstructs are provided, comprising a promoter which is capable ofinitiating the synthesis of viral RNA from cDNA followed by a 5′sequence which is capable of initiating transcription of an alphavirus,a nucleotide sequence encoding alphavirus non-structural proteins, afirst viral junction region which has been inactivated such that viraltranscription of the subgenomic fragment is prevented, followed by asecond viral junction region which is active, or which has been modifiedsuch that viral transcription of the subgenomic fragment is reduced, analphavirus RNA polymerase recognition sequence, and a 3′ sequence whichcontrols transcription termination.

Within other aspects of the present invention, eukaryotic layered vectorinitiation systems are provided which are capable of expressing aheterologous nucleic acid sequence in a eukaryotic cell transformed ortransfected therewith. In particular embodiments, eukaryotic layeredvector initiation systems are provided, comprising a promoter which iscapable of initiating the 5′ synthesis of RNA from cDNA, a vectorconstruct which is capable of autonomous replication in a cell, thevector construct being capable of expressing a heterologous nucleic acidsequence, and a 3′ sequence which controls transcription termination.

Within a related aspect, eukaryotic layered vector initiation systemsare provided, comprising a DNA promoter which is capable of initiatingthe 5′ synthesis of RNA from cDNA, a vector construct which is capableof autonomous replication in a cell, the vector construct being capableof expressing a heterologous ribonucleic acid sequence, and a 3′ DNAsequence which controls transcription termination.

Within one embodiment, the vector construct within the eukaryoticlayered vector initiation systems of the present invention is analphavirus vector construct. Within other embodiments, the construct isderived from a virus selected from the group consisting of poliovirus,rhinovirus, coxsackieviruses, rubella, yellow fever, HCV, TGEV, IBV,MHV, BCV, parainfluenza virus, mumps virus, measles virus, respiratorysyncytial virus, influenza virus, RSV, MoMLV, HIV, HTLV, hepatitis deltavirus and Astrovirus. Within yet other embodiments, the promoter whichis capable of initiating the 5′ synthesis of RNA from cDNA is selectedfrom the group consisting of the MoMLV promoter, metallothioneinpromoter, glucocorticoid promoter, SV 40 promoter, and the CMV promoter.Within further embodiments, the eukaryotic layered vector initiationsystems further comprise a polyadenylation sequence.

In further embodiments of the invention, in any of the above aspects,the vectors (e.g., alphavirus vector construct, alphavirus cDNA vectorconstruct, or eukaryotic layered vector initiation system) may bederived from an alphavirus selected from the group consisting of Aura,Fort Morgan, Venezuelan Equine Encephalitis, Ross River, Semliki Forest,Sindbis, and Mayaro.

In other embodiments, the vectors described above contain a heterologoussequence. Typically, such vectors contain a heterologous nucleotidesequence of greater than 100 bases, generally the heterologousnucleotide sequence is greater than 3 kb, and sometimes greater than 5kb, or even 8 kb. In various embodiments, the heterologous sequence is asequence encoding a protein selected from the group consisting of IL-1,IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12,IL-13, IL-14, IL-15,alpha-, beta-, or gamma-IFN, G-CSF, and GM-CSF.Within other embodiments of the invention, the heterologous sequence mayencode a lymphokine receptor. Representative examples of such receptorsinclude receptors for any of the lymphokines set forth above.

In still other embodiments, the vectors described above include aselected heterologous sequence which may be obtained from a virusselected from the group consisting of influenza virus, HPV, HBV, HCV,EBV, HIV, HSV, FeLV, FIV, Hanta virus, HTLV I, HTLV II and CMV. Withinone preferred embodiment, the heterologous sequence obtained from HPVencodes a protein selected from the group consisting of E5,E6, E7 andL1. In yet other embodiments, the vectors described above include aselected heterologous sequence encoding an HIV protein selected from thegroup consisting of HIV gp120 and gag.

The selected heterologous sequences described above also may be anantisense sequence, noncoding sense sequence, or ribozyme sequence. Inpreferred embodiments, the antisense or noncoding sense sequence isselected from the group consisting of sequences which are complementaryto influenza virus, HPV, HBV, HCV, EBV, HIV, HSV, FeLV, FIV, Hantavirus, HTLV I, HTLV II, and CMV sequences.

In another embodiment, the vectors described above contain no alphavirusstructural protein genes. Within other embodiments, the selectedheterologous sequence is located downstream from a viral junctionregion. In the vectors described above having a second viral junction,the selected heterologous sequence may, within certain embodiments, belocated downstream from the second viral junction region. Where theheterologous sequence is located downstream from a viral junctionregion, the vector construct may further comprise a polylinker locatedsubsequent to the viral junction region. Within preferred embodiments,such polylinkers do not contain a restriction endonuclease recognitionsequence present in the wild-type alphavirus sequence.

In yet another embodiment, in the vectors described above the selectedheterologous sequence may be located within the nucleotide sequenceencoding alphavirus non-structural proteins.

In particular embodiments, the vectors described above include a viraljunction region consisting of the nucleotide sequence as shown in FIG.3, from nucleotide number 7579, to nucleotide number 7597 (SEQ. ID NO.1). In alternative embodiments, where the vector includes a second viraljunction, an E3 adenovirus gene may be located downstream from thesecond viral junction region. Vectors of the present invention may alsofurther comprise a non-alphavirus (for example retrovirus, coronavirus,hepatitis B virus) packaging sequence located between the first viraljunction region and the second viral junction region, or in thenonstructural protein coding region.

In further aspects, the present invention provides an isolatedrecombinant alphavirus vector which does not contain a functional viraljunction region, and which in preferred embodiments produces reducedviral transcription of the subgenomic fragment.

In still a further aspect, the present invention provides an alphavirusstructural protein expression cassette, comprising a promoter and one ormore alphavirus structural protein genes, the promoter being capable ofdirecting the expression of alphavirus structural proteins. In variousembodiments, the expression cassette is capable of expressing alphavirusstructural proteins, such as an alphavirus structural protein selectedfrom the group consisting of C, 6K, E3, E2, and E1.

Within other embodiments, the alphavirus structural protein is derivedfrom an alphavirus selected from the group consisting of Aura, FortMorgan, Venezuelan Equine Encephalitis, Ross River, Semliki Forest,Sindbis and Mayaro viruses.

In yet another aspect, the present invention provides an alphavirusstructural protein expression cassette, comprising a promoter, one ormore alphavirus structural proteins, and a heterologous ligand sequence,the promoter being capable of directing the expression of the alphavirusstructural proteins and the heterologous sequence. In variousembodiments, the heterologous ligand sequence is selected from the groupconsisting of VSVG, HIV gp120, antibody, insulin, and CD4.

In certain embodiments, the expression cassettes described above includea promoter selected from the group consisting of metallothionein,Drosophila actin 5C distal, SV40, heat shock protein 65, heat shockprotein 70, Py, RSV, BK, JC, MuLV, MMTV, alphavirus junction region CMVand VA1RNA.

The present invention also provides packaging cell lines and producercell lines suitable for producing recombinant alphavirus particles. Suchpackaging or producer cell lines may be either mammalian ornon-mammalian (e.g., insect cells, such as mosquito cells). In certainembodiments, the packaging cell lines and producer cell lines contain anintegrated alphavirus structural protein expression cassette.

Within one embodiment, packaging cell lines are provided which, uponintroduction of a vector construct, produce alphavirus particles capableof infecting human cells. Within other embodiments, the packaging cellline produces alphavirus particles in response to one or more factors.Within certain embodiments, an alphavirus inhibitory protein is notproduced within the packaging cell line.

Within other aspects, retroviral-derived packaging cell lines areprovided which are suitable for packaging and production of analphavirus vector. Within one embodiment, a retroviral-derived producercell line suitable for packaging and production of an alphavirus vectoris provided, comprising an expression cassette which directs theexpression of gag/pol, an expression cassette which directs theexpression of env, and alphavirus vector construct containing aretroviral packaging sequence.

Within another aspect, HBV-derived and coronavirus-derived packagingcell lines are provided which are suitable for packaging and productionof and alphavirus vector. Within one embodiment, an HBV-derivedpackaging cell line is provided, comprising an expression cassette whichdirects the expression of HBV core, PreS/S, and P proteins. Withinanother embodiment, a coronavirus-derived packaging cell line isprovided, comprising an expression cassette which directs the expressionof coronavirus N, M, and S proteins.

Within another aspect, a VSV-G derived packaging cell is provided whichis suitable for packaging and production of an alphavirus vector,comprising a stably integrated expression cassette which directs theexpression of VSV-G. Within a further embodiment, such packaging celllines comprise a stably integrated expression cassette which directs theexpression of one or more alphavirus structural proteins.

Within yet other aspects, producer cell lines are provided based uponthe above packaging cell lines. Within one embodiment, such producercell lines produce recombinant alphavirus particles in response to adifferentiation state of the producer cell line. Within otherembodiments, such producer cell lines produce recombinant alphavirusparticles in response to one or more factors.

As utilized with the context of the present invention, alphavirusproducer cell line refers to a cell line which is capable of producingrecombinant alphavirus particles. The producer cell line should includean integrated alphavirus structural protein expression cassette capableof directing the expression of alphavirus structural protein(s), andalso, an alphavirus vector construct. Preferably, the alphavirus vectorconstruct is a cDNA vector construct. More preferably, the alphavirusvector construct is an integrated cDNA vector construct. When thealphavirus construct is an integrated cDNA vector construct, it may, insome instances, function only in response to one or more factors, or thedifferentiation state of the alphavirus producer cell line.

In still yet another aspect, the present invention provides alphavirusparticles which, upon introduction into a BHK cell, produces an infectedcell which is viable at least 24 hours and as much as 48, 72, or 96hours, or 1 week after infection. Also provided are mammalian cellswhich contain such alphavirus particles. In addition, recombinantalphavirus particles capable of infecting human cells are provided.

In another aspect, the present invention provides recombinant alphavirusparticles which, upon introduction into a BHK cell, produces an infectedcell which is viable at least 24 hours after infection, the particlealso carrying a vector construct which directs the expression of atleast one antigen or modified form thereof in target cells infected withthe alphavirus particle, the antigen or modified form thereof beingcapable of stimulating an immune response within an animal. In variousembodiments, the expressed antigen or modified form thereof elicits acell-mediated immune response, preferably an HLA class I-restrictedimmune response.

In still another aspect, the present invention provides recombinantalphavirus particles which carry a vector capable of directing theexpression of a palliative in cells infected with the alphavirusparticle, the palliative being capable of inhibiting a function of apathogenic agent necessary for pathogenicity. In various embodiments,the pathogenic agent is a virus, fungi, protozoa, or bacteria, and theinhibited function is selected from the group consisting of adsorption,replication, gene expression, assembly, and exit of the pathogenic agentfrom infected cells. In other embodiments, the pathogenic agent is acancerous cell, cancer-promoting growth factor, autoimmune disorder,cardiovascular disorders such as restenosis, osteoporosis and malepattern baldness, and the inhibited function selected from the groupconsisting of cell viability and cell replication. In furtherembodiments, the vector directs the expression of a toxic palliative ininfected target cells in response to the presence in such cells of anentity associated with the pathogenic agent; preferably the palliativeis capable of selectively inhibiting the expression of a pathogenic geneor inhibiting the activity of a protein produced by the pathogenicagent. In still further embodiments, the palliative comprises aninhibiting peptide specific for viral protease, an antisense RNAcomplementary to RNA sequences necessary for pathogenicity, a sense RNAcomplementary to RNA sequences necessary for pathogenicity, or adefective structural protein of a pathogenic agent, such protein beingcapable of inhibiting assembly of the pathogenic agent.

In yet further embodiments, recombinant alphavirus particles describedabove direct the expression of a palliative, more particularly, directthe expression of a gene product capable of activating an otherwiseinactive precursor into an active inhibitor of the pathogenic agent, forexample, the herpes thymidine kinase gene product, a tumor suppressorgene, or a protein that activates a compound with little or nocytotoxicity into a toxic product in the presence of a pathogenic agent,thereby effecting localized therapy to the pathogenic agent.Alternatively, the recombinant alphavirus particle directs theexpression of a protein that is toxic upon processing or modification bya protein derived from a pathogenic agent, a reporting product on thesurface of target cells infected with the alphavirus and containing thepathogenic agent, or an RNA molecule which functions as an antisense orribozyme specific for a pathogenic RNA molecule required for pathogens.In certain embodiments, in the alphavirus particles described above, theprotein is herpes thymidine kinase or CD4.

In yet further aspects, the present invention provides recombinantalphavirus particles which direct the expression of a gene capable ofsuppressing one or more elements of the immune system in target cellsinfected with the alphavirus vector, and an alphavirus particle whichdirects the expression of a blocking element in cells infected with thealphavirus vector, the blocking element being capable of binding toeither a receptor or an agent such that the receptor/agent interactionis blocked.

In further aspects, methods are provided for administering any of theabove-described recombinant alphavirus particles or vectors, for aprophylactic or therapeutic effect. For example, within one aspect, thepresent invention provides methods of stimulating an immune response toan antigen, comprising the step of infecting susceptible target cellswith a recombinant alphavirus particle which directs the expression ofat least one antigen or modified form thereof in target cells infectedwith the alphavirus, the antigen or modified form thereof being capableof stimulating an immune response within an animal. In one embodiment,the target cells are infected in vivo, although within other embodimentsthe target cells are removed, infected ex vivo, and returned to theanimal.

In still further aspects of the present invention, methods ofstimulating an immune response to a pathogenic antigen are provided,comprising the step of infecting susceptible target cells with arecombinant alphavirus particle which directs the expression of amodified form of a pathogenic antigen in target cells infected with thealphavirus, the modified antigen being capable of stimulating an immuneresponse within an animal but having reduced pathogenicity relative tothe pathogenic antigen.

In even further aspects of the present invention, methods of stimulatingan immune response to an antigen are provided, comprising infectingsusceptible target cells with a recombinant alphavirus particle whichdirects the expression of a peptide having multiple epitopes, one ormore of the epitopes derived from different proteins.

In yet another aspect of the invention, methods of stimulating an immuneresponse within a warm-blooded animal are provided, comprising infectingsusceptible target cells associated with a warm-blooded animal withnucleic acid sequences coding for either individual class I or class IIMHC protein, or combinations thereof, and infecting the cells with analphavirus particle which directs the expression of at least one antigenor modified form thereof in target cells infected with the alphavirusparticle, the antigen or modified form thereof being capable ofstimulating an immune response within the animal.

In another aspect of the present invention, methods of inhibiting apathogenic agent are provided, comprising infecting susceptible targetcells with an alphavirus particle which directs the expression of apalliative in cells infected with the alphavirus particle, thepalliative being capable of inhibiting a function of a pathogenic agentnecessary for pathogenicity.

As utilized within the context of the present invention, vector orvector constructs which direct the expression of a heterologous sequenceof interest in fact refers to the transcribed vector RNA, which directsthe expression of the heterologous sequence of interest. In addition,although “animals” are generally referred to, it should be understoodthat the present invention may be readily applied to a wide variety ofanimals (both mammalian and non-mammalian), including for example,humans, chimps, macaques, cows, horses, sheep, dogs, birds, cats, fish,rats, and mice. Further, although alphaviruses such as Sindbis may bespecifically described herein, it should be understood that a widevariety of other alphaviruses may also be utilized including, forexample, Aura, Venezuelan Equine Encephalitis, Fort Morgan, Ross River,Semliki Forest, and Mayaro.

Within other aspects of the present invention, methods are provided fordelivering a heterologous nucleic acid sequence to an animal comprisingthe steps of administering to the warm-blooded animal a eukaryoticlayered vector initiation system as described above. Within certainembodiments, the eukaryotic layered vector initiation system may beintroduced into the target cells directly as a DNA molecule by physicalmeans, as a complex with various liposome formulations, or as aDNA-ligand complex including the vector molecule (e.g., along with apolycation compound such as polylysine, a receptor specific ligand, or apsoralen inactivated virus such as Sendai or Adenovirus).

Within yet other aspects of the invention, ex vivo cells are infectedwith any of the above-described recombinant alphaviruses are provided.Within yet other aspects, recombinant alphavirus particles are providedwhich are resistant to inactivation in serum. As utilized herein,recombinant alphavirus particles are considered to be resistant toinactivation in serum if the ratio of surviving particles toinput/starting particles in a complement inactivation assay is greaterin a statistically significant manner, preferably at least 5-fold, andas much as 10- to 20- fold, as compared to a reference sample producedin BHK cells. Within further aspects, pharmaceutical compositions areprovided comprising any of the above-described vectors, or recombinantalphavirus particles, in combination with a physiologically acceptablecarrier or diluent.

In yet another aspect of the invention, the eukaryotic layered vectorinitiation systems provided enable new methods for large scalerecombinant protein expression.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings. In addition, various references are set forth below whichdescribe in more detail certain procedures or compositions (e.g,plasmids, etc.). These references are incorporated herein by referencein their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of Sindbis virus genomicorganization.

FIG. 2 is an illustration which depicts a method for amplification of aSindbis RNA genome by RT-PCR.

FIGS. 3A-H set forth the sequence of a representative Eukaryotic LayeredVector Initiation System derived from Sindbis (SEQ. ID NO. 1).

FIG. 4 is a schematic illustration of a Sindbis Basic Vector and aSindbis-luciferase Vector.

FIG. 5 is an illustration of Sindbis Helper Vector Construction.

FIG. 6 is a graph which illustrates expression and rescue of aSindbis-luciferase Vector.

FIG. 7 is an illustration of one method for modifying a Sindbis junctionregion.

FIG. 8 is a schematic illustration of a representative embodiment of aEukaryotic Layered Vector Initiation System.

FIG. 9 is a graph which shows a time course for luciferase expressionfrom ELVIS-LUC and SINBV-LUC vectors.

FIG. 10 is a bar graph which depicts the level of ELVIS vector reportergene expression compared to several different vector constructs.

FIG. 11 is a schematic illustration of Sindbis Packaging ExpressionCassettes.

FIG. 12 is a bar graph which shows SIN-luc vector packaging byrepresentative packaging cell lines.

FIG. 13 is a bar graph which shows SIN-luc vector packaging by PCL clone#18 over time.

FIG. 14 is a bar graph which depicts the level of expression by severaldifferent luciferase vectors in BHK cells and undifferentiated F9 cells.

FIG. 15 is a schematic illustration of how Astroviruses or otherheterologous viruses may be used to express Sindbis structural proteins.

FIG. 16A is a bar graph which shows Sindbis BV-HBe expression andpackaging in BHK cells (lysate). FIG. 16B is a bar graph which showsSindbis BV-HBe expression and packaging in BHK cells (supernatant).

FIG. 17 is a bar graph which shows Sindbis BV-HB core expression andpackaging in BHK cells.

FIG. 18 is a bar graph which shows a comparison of HB core expressedfrom Sindbis and RETROVECTORS™.

FIG. 19 is a bar graph which shows ELVIS-HBe vector expression in BHKcells.

FIGS. 20A-20D are is a schematic illustration of several representativemechanisms for activating a disabled viral junction region by “RNAloop-out.”

FIG. 21 is a western blot demonstrating expression of capsid proteinafter transfection with pMAM/C, selection in HAT media, and inductionwith dexamethasone.

FIGS. 22A-22B depict is a bar graph which demonstrates the level ofexpression of luciferase in BHK cells transfected with ELVIS-LUC vector,and various modifications thereof.

FIG. 23 is a bar graph which demonstrates the level of luciferase or β-galactosidase expression in BHK cells transfected with ELVIS expressionvectors, co-transfected with ELVIS expression and helper vectors, ortransduced with packaged ELVIS expression vectors.

FIG. 24 depicts a photomicrograph of a ELVIS-β-gal injected rat muscleat three days post inoculation. A transverse cryosection fromgastronemius muscle injected with 50 μg of ELVIS-β-gal contained in PBSis shown. Four blue stained transverse fibers are evident.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention, it may be helpful to anunderstanding thereof to first set forth definitions of certain termsthat will be used hereinafter.

“Alphavirus vector construct” refers to an assembly which is capable ofdirecting the expression of a sequence(s) of gene(s) of interest. Thevector construct should include a 5′ sequence which is capable ofinitiating transcription of an alphavirus, as well as sequence(s) which,when expressed, code for biologically active alphavirus non-structuralproteins (e.g., NSP1, NSP3, and NSP4), and an alphavirus RNA polymeraserecognition sequence. In addition, the vector construct should include aviral junction region which may, in certain embodiments, be modified inorder to prevent, increase, or reduce viral transcription of thesubgenomic fragment, and an alphavirus RNA polymerase recognitionsequence. The vector may also include nucleic acid molecule(s) which areof a size sufficient to allow production of viable virus, a 5′ promoterwhich is capable of initiating the synthesis of viral RNA in vitro fromcDNA, as well as one or more restriction sites, and a polyadenylationsequence.

“Alphavirus cDNA vector construct” refers to an assembly which iscapable of directing the expression of a sequence(s) or gene(s) ofinterest. The vector construct should include a 5′ sequence which iscapable of initiating transcription of an alphavirus, as well assequence(s) which, when expressed, code for biologically activealphavirus non-structural proteins (e.g., NSP1, NSP2, NSP3, and NSP4),and an alphavirus RNA polymerase recognition sequence. In addition, thevector construct should include a 5′ promoter which is capable ofinitiating the synthesis of viral RNA from cDNA, a viral junction regionwhich may, in certain embodiments, be modified in order to prevent,increase, or reduce viral transcription of the subgenomic fragment, analphavirus RNA polymerase recognition sequence, and a 3′ sequence whichcontrols transcription termination. The vector may also include nucleicacid molecule(s) which are of a size sufficient to allow production ofviable virus, splice recognition sequences, a catalytic ribozymeprocessing sequence, as well as a polyadenylation sequence.

“Expression cassette” refers to a recombinantly produced nucleic acidmolecule which is capable of directing the expression of one or moreproteins. The expression cassette must include a promoter capable ofdirecting the expression of said proteins, and a sequence encoding oneor more proteins, said proteins preferably comprising alphavirusstructural protein(s). Optionally the expression cassette may includetranscription termination, splice recognition, and polyadenylationaddition sites. Preferred promoters include the CMV, MMTV, MoMLV, andadenovirus VA1RNA promoters. In addition, the expression cassette maycontain selectable markers such as Neo, SV2 Neo, hygromycin, phleomycin,histidinol, and DHFR.

“Alphavirus producer cell line” refers to a cell line which is capableof producing recombinant alphavirus particles. The producer cell lineshould include an integrated alphavirus structural protein expressioncassette capable of directing the expression of alphavirus structuralprotein(s), and also, an alphavirus vector construct. Preferably, thealphavirus vector construct is a cDNA vector construct. More preferably,the alphavirus vector construct is an integrated cDNA vector construct.When the alphavirus vector construct is an integrated cDNA vectorconstruct, it may, in some instances, function only in response to oneor more factors, or the differentiation state of the alphavirus producercell line.

“Recombinant alphavirus particle” refers to a capsid which contains analphavirus vector construct. Preferably, the capsid is an alphaviruscapsid and is contained within a lipid bilayer, such as a cell membrane,in which viral-encoded proteins are embedded. In some instances, thealphavirus vector construct may be contained in a capsid derived fromviruses other than alphaviruses (for example, retroviruses,coronaviruses, and hepatitis B virus). A variety of alphavirus vectorsmay be contained within the recombinant alphavirus particle, includingthe alphavirus vector constructs of the present invention.

A. SOURCES OF ALPHAVIRUS

As noted above, the present invention provides alphavirus vectorconstructs, alphavirus particles containing such constructs, as well asmethods for utilizing such vector constructs and particles. Briefly,sequences encoding wild-type alphavirus suitable for use in preparingthe above-described vector constructs and particles may be readilyobtained given the disclosure provided herein from naturally-occurringsources, or from depositories (e.g., the American Type CultureCollection, Rockville, Md.).

Representative examples of suitable alphaviruses include Aura (ATCCVR-368), Bebaru virus (ATCC VR-600, ATCC VR-1240), Cabassou (ATCCVR-922), Chikungunya virus (ATCC VR-64, ATCC VR-1241), Eastern equineencephalomyelitis virus (ATCC VR-65, ATCC VR-1242), Fort Morgan (ATCCVR-924), Getah virus (ATCC VR-369, ATCC VR-1243), Kyzylagach (ATCCVR-927), Mayaro (ATCC VR-66), Mayaro virus (ATCC VR-1277), Middleburg(ATCC VR-370), Mucambo virus (ATCC VR-580, ATCC VR-1244), Ndumu (ATCCVR-371), Pixuna virus (ATCC VR-372, ATCC VR-1245), Ross River virus(ATCC VR-373,ATCC VR-1246), Semliki Forest (ATCC VR-67, ATCC VR-1247),Sindbis virus (ATCC VR-68,ATCC VR-1248), Tonate (ATCC VR-925), Triniti(ATCC VR-469), UNA (ATCC VR-374), Venezuelan equine encephalomyelitis(ATCC VR-69), Venezuelan equine encephalomyelitis virus (ATCC VR-923,ATCC VR-1250 ATCC VR-1249, ATCC VR-532), Western equineencephalomyelitis (ATCC VR-70, ATCC VR-1251, ATCC VR-622, ATCC VR-1252),Whataroa (ATCC VR-926), and Y-62-33 (ATCC VR-375).

B. SEQUENCES WHICH ENCODE WILD-TYPE SINDBIS VIRUS

Within one particularly preferred aspect of the present invention, thesequences which encode wild-type alphavirus may be obtained from Sindbisvirus. In particular, within one embodiment of the invention (and asdescribed in more detail below in Example 1), a Sindbis full-lengthgenomic cDNA clone may be obtained by linking the 5′ end of a Sindbisvirus cDNA clone to a bacteriophage RNA polymerase promoter, and the 3′end of the cDNA clone to a polyadenosine (poly A) tract of at least 25nucleotides. In particular, synthesis of the first cDNA strand from theviral RNA template may be accomplished with a 3′ oligonucleotide primerhaving a consecutive sequence comprising an enzyme recognition sequence,a sequence of 25 deoxythmidine nucleotides, and a stretch ofapproximately 18 nucleotides which is complementary to the viral 3′ end,and with a 5′ primer containing buffer nucleotides, an enzymerecognition sequence, a bacteriophage promoter, and a sequencecomplimentary to the viral 5′ end. The enzyme recognition sites presenton each of these primers should be different from each other, and notfound in the Sindbis virus. Further, the first nucleotide linked to the3′ end of the bacteriophage RNA polymerase promoter may be the authenticfirst nucleotide of the RNA virus, or may contain one or more additionalnon-viral nucleotides. RNA transcribed in vitro from the viral cDNAclone, having the construction described above and linearized bydigestion with the unique dT:dA 3′ distal restriction enzyme will, afterintroduction into the appropriate eukaryotic cell, initiate the sameinfection cycle which is characteristic of infection by the wild-typevirus from which the cDNA was cloned. This viral cDNA clone, whichyields RNA able to initiate infection after in vitro transcription, isreferred to below as an “infectious cDNA clone.”

C. PRODUCTION OF RECOMBINANT ALPHAVIRUS VECTOR CONSTRUCTS WITHINACTIVATED VIRAL JUNCTION REGIONS

An infectious cDNA clone prepared as described above (or utilizingsequences encoding an alphavirus obtained from other sources) may bereadily utilized to prepare alphavirus vector constructs of the presentinvention. Briefly, within one aspect of the present invention,recombinant alphavirus vector constructs are provided, comprising a 5′sequence which is capable of initiating transcription of an alphavirus,a nucleotide sequence encoding alphavirus nonstructural proteins, aviral junction region which has been inactivated such that viraltranscription of the subgenomic fragment is prevented, and an alphavirusRNA polymerase recognition sequence. As will be discussed in greaterdetail below, alphavirus vector constructs which have inactivated viraljunction regions do not transcribe the subgenomic fragment, making themsuitable for a wide variety of applications.

1. RNA POLYMERASE PROMOTER

As noted above, within certain embodiments of the invention alphavirusvector constructs are provided which contain a 5′ promoter which iscapable of initiating the synthesis of viral RNA in vitro from cDNA.Particularly, preferred 5′ promoters include both eukaryotic andprokaryotic promoters, such as, for example, the β-galactosidasepromoter, trpE promoter, lacZ promoter, T7 promoter, T3 promoter, SP6promoter, SV40 promoter, CMV promoter, and MoMLV LTR.

2. SEQUENCES WHICH INITIATE TRANSCRIPTION

As noted above, within preferred embodiments the alphavirus vectorconstructs of the present invention contain a 5′ sequence which iscapable of initiating transcription of an alphavirus. Representativeexamples of such sequences include nucleotides 1-60, and to a lesserextent nucleotides 150-210, of the wild-type Sindbis virus (see FIG. 3),nucleotides 10-75 for tRNA Asparagine (Schlesinger et al., U.S. Pat. No.5,091,309), and 5′ sequences from other Togaviruses which initiatetranscription.

3. ALPHAVIRUS NONSTRUCTURAL PROTEINS

Alphavirus vector constructs of the present invention should alsocontain sequences which encode alphavirus nonstructural proteins (NSPs).As an example, for Sindbis virus there are four nonstructural proteinsNSP1, NSP2, NSP3 and NSP4, which encode proteins that enable the virusto self-replicate. Nonstructural proteins 1 through 3 (NSP1-NSP3) are,within one embodiment of the invention, encoded by nucleotides 60 to5750 of the wild-type Sindbis virus (see FIG. 3). These proteins areproduced as a polyprotein and later cleaved into nonstructural proteinsNSP1, NSP2, and NSP3. NSP4 is, within one embodiment, encoded bynucleotides 5928 to 7579 (see FIG. 3).

It will be evident to one of ordinary skill in the art that a widevariety of sequences which encode alphavirus nonstructural proteins, inaddition to those discussed above, may be utilized in the presentinvention, and are therefore deemed to fall within the scope of thephase “Alphavirus Nonstructural Proteins.” For example, within oneembodiment of the invention, due to the degeneracy of the genetic code,more than one codon may code for a given amino acid. Therefore, a widevariety of nucleic acid sequences which encode alphavirus nonstructuralproteins may be generated. Within other embodiments of the invention, avariety of other nonstructural protein derivatives may be made,including for example, various substitutions, insertions, or deletions,the net result of which do not alter the biological activity of thealphavirus nonstructural proteins. Within the context of the presentinvention, alphavirus nonstructural proteins are deemed to be“biologically active” in toto if they promote the self-replication ofthe vector construct. Self-replication, which refers to replication ofviral nucleic acids and not the production of infectious virus, may bereadily determined by metabolic labelling or RNase protection assaysperformed over a course of time. Methods for making such derivatives maybe readily accomplished by one of ordinary skill in the art given thedisclosure provided herein (see also, Molecular Cloning: A LaboratoryManual (2nd ed.), Cold Spring Harbor Laboratory Press).

4. VIRAL JUNCTION REGIONS

Within this aspect of the invention, the alphavirus vector constructsmay also include a viral junction region which has been inactivated,such that viral transcription of the subgenomic fragment is prevented.Briefly, the alphavirus viral junction region normally controlstranscription initiation of the subgenomic mRNA. In the case of theSindbis virus, the normal viral junction region typically begins atapproximately nucleotide number 7579 and continues through at leastnucleotide number 7612 (and possibly beyond). At a minimum, nucleotides7579 to 7602 (5′ - ATC TCT ACG GTG GTC CTA AAT AGT-SEQ. ID NO. 2) arebelieved necessary for transcription of the subgenomic fragment. Thisregion (nucleotides 7579 to 7602) is hereinafter referred to as the“minimal junction region core.”

Within preferred embodiments of the invention (and as described in moredetail below), the viral junction region is inactivated in order toprevent viral transcription of the subgenomic fragment. As utilizedwithin the context of the present invention, “inactivated” means thatthe fragment corresponding to the initiation point of the subgenomicfragment, as measured by a RNase protection assay, is not detected.(Representative assays are described by Melton et al., Nuc. Acids Res.12:7035-7056, 1984; Calzon et al., Methods in Enz. 152:611-632, 1987;and Kekule et al., Nature 343:457-461, 1990.)

Within one embodiment of the invention, the viral junction region isinactivated by truncating the viral junction region at nucleotide 7597(i.e., the viral junction region will then consist of the sequence asshown in FIG. 3, from nucleotide 7579 to nucleotide 7597). Thistruncation prevents transcription of the subgenomic fragment, andadditionally permits synthesis of the complete NSP4 region (which isencoded by nucleotides 5928 to 7579).

As will be evident to one of ordinary skill in the art given thedisclosure provided herein, a wide variety of other deletionssubstitutions or insertions may also be made in order to inactivate theviral junction region. For example, within other embodiments of theinvention the viral junction region may be further truncated into theregion which encodes NSP4, thereby preventing viral transcription fromthe subgenomic fragment while retaining the biological activity of NSP4.Alternatively, within other embodiments, due to the redundancy of thegenetic code, nucleotide substitutions may be made in the sequenceencoding NSP4, the net effect of which does not alter the biologicalactivity of NSP4 yet, nevertheless, prevents transcription of thesubgenomic fragment.

5. ALPHAVIRUS RNA POLYMER RECOGNITION SEQUENCE, AND POLY-A TAIL

As noted above, alphavirus vector constructs of the present inventionshould also include an alphavirus RNA polymerase recognition sequence(also termed “alphavirus replicase recognition sequence”). Briefly, thealphavirus RNA polymerase recognition sequence provides a recognitionsite at which the virus begins replication by synthesis of the negativestrand. A wide variety of sequences may be utilized as an alphavirus RNApolymerase recognition sequence. For example, within one embodiment,Sindbis vector constructs of the present invention include a Sindbispolymerase recognition sequence which is encoded by nucleotides 11,647to 11,703 (see FIG. 3). Within other embodiments, the Sindbis polymeraserecognition is truncated to the smallest region which can still functionas a recognition sequence (e.g., nucleotides 11,684 to 11,703 of FIG.3).

Within preferred embodiments of the invention, the vector construct mayadditionally contain a polyA tail. Briefly, the polyA tail may be of anysize which is sufficient to promote stability in the cytoplasm, therebyincreasing the efficiency of initiating the viral life cycle. Withinvarious embodiments of the invention, the polyA tail comprises at least10 adenosine nucleotides, and most preferably, at least 25 nucleotides.

D. OTHER ALPHAVIRUS VECTOR CONSTRUCTS

In addition to the vector constructs which are generally describedabove, a wide variety of other alphavirus vector constructs may also beprepared utilizing the disclosure provided herein.

1. MODIFIED VIRAL JUNCTION REGIONS

As noted above, the present invention provides viral junction regionswhich have been modified from the wild-type sequence. Within the contextof the present invention, modified viral junction regions should beunderstood to include junction regions which have wild-type activity,but a non-wild-type sequence, as well as junction regions withincreased, decreased, or no activity. For example, within one aspect ofthe invention, alphavirus vector constructs are provided wherein theviral junction region has been modified, such that viral transcriptionof the subgenomic fragment is reduced. Briefly, infection of cells withwild-type alphavirus normally results in cell death as a result ofabundant viral transcription of the subgenomic fragment initiated fromthe viral junction region. This large abundance of RNA molecules canoverwhelm the transcriptional machinery of the infected cell, ultimatelyresulting in death of the cell. In applications where it is desired thatinfection of a target cell should result in a therapeutic effect (e.g.,strand scission of a target nucleic acid or prolonged expression of aheterologous protein) rather than cell death, several modifications tothe alphavirus vector construct (in addition to inactivating the vectorconstruct, as described above) may be made in order to reduce the levelof viral transcription of the subgenomic fragment, and thereby prolongthe life of the vector infected target cell. Within the context of thepresent invention, viral transcription of the subgenomic fragment isconsidered to be “reduced” if it produces less subgenomic fragment thana standard wild-type alphavirus (e.g., Sindbis virus ATCC No. VR-1248)as determined by a RNase protection assay.

Viral junction regions may be modified by a variety of methods in orderto reduce the level of viral transcription of the subgenomic fragment.For example, within one embodiment of the invention, due to theredundancy of the genetic code nucleotide substitutions may be made inthe viral junction region 7579 to 7597, the net effect of which does notalter the amino acid sequence NSP4 (or, within other embodiments, thebiological activity of NSP4), and yet reduces the level of viraltranscription of the subgenomic fragment. If the modified vectorconstruct includes nucleotides beyond 7597 (e.g., to 7602 or 7612),further nucleotide substitutions may likewise be made, although, sinceNSP4 terminates at 7597, such substitutions need not be based upongenetic redundancy. Representative examples of modified viral junctionregions are described in more detail below in Example 3.

2. TANDEM VIRAL JUNCTION REGIONS

Within other aspects of the invention, alphavirus vector constructs areprovided, with comprise a 5′ sequence which is capable of initiatingtranscription of an alphavirus, a nucleotide sequence encodingalphavirus non-structural proteins, a first viral junction region whichhas been inactivated such that viral transcription of the subgenomicfragment is prevented, a second viral junction region which is active,or which has been modified such that viral transcription of thesubgenomic fragment is reduced, and an alphavirus RNA polymeraserecognition sequence. Such vector constructs are referred to as “tandem”vector constructs because they comprise a first inactivated (or“disabled”) viral junction region, as well as a second modified(“synthetic”) or unmodified viral junction region. Within preferredembodiments of the invention, the inactivated junction region isfollowed directly by the second viral junction region.

In applications where a low level of subgenomic transcription isrequired, a minimal junction region core may be inserted downstream intandem to the inactivated junction region. In order to graduallyincrease the level of subgenomic transcription for the desired effect,sequences corresponding to the entire junction region may be added tothe in-tandem junction region, in increments.

3. THE ADENOVIRUS E3 GENE

Within another aspect of the invention, an adenovirus E3 gene isinserted into a tandem vector construct following the second viraljunction region, in order to down-regulate HLA expression in alphavirusinfected cells. Briefly, within various embodiments of the invention,repeated inoculations of a gene therapeutic into the same individual isdesirable. However, repeated inoculations of alphaviruses such as theSindbis virus may lead to the development of specific antibodies orcell-mediated immune response against Sindbis viral nonstructuralproteins (NSPs). Thus, it may be necessary to mitigate the host immuneresponse targeted to vector-specific proteins in order to administerrepeated doses to the same individual.

Therefore, within one embodiment of the invention, products of theAdenovirus type 2 early region gene 3 are utilized in order todown-regulate the expression of integral histocompatibility antigensexpressed on the surface of infected cells. Briefly, the E3 19,000dalton (E3/19K) protein binds to, and forms a molecular complex with,class I H-2/HLA antigens in the endoplasmic reticulum, preventingterminal glycosylation pathways necessary for the full maturation andsubsequent transport of the class I H-2/HLA antigens to the cellmembrane. In target cells infected with an alphavirus vector encodingthe Ad 2 E3 protein, co-expression of the viral nonstructural proteinsin the context of class I antigens will not occur. Thus, it is possibleto administer repeated doses of an alphavirus vector which expresses theAd 2 E3 protein as a component of its therapeutic palliative to the sameindividual. A representative example of the use of the Adenovirus E3gene is set forth in more detail below in Example 4A.

4. THE CMV H301 GENE

Other methods may also be utilized in order to mitigate a host's immuneresponse against viral NSPs. For example, within another aspect of theinvention, the human cytomegalovirus (“HCMV”) H301 gene is cloned intoan alphavirus vector construct, preferably immediately following thesecond viral junction region in a tandem vector, in order to inhibithost CTL response directed against viral specific proteins expressed invector infected cells.

Briefly, 2-Microglobulin (2 m) protein binds to the 1, 2 and 3 domainsof the alpha-chain of the class I major histocompatibility molecules ofhigher eukaryotes. Preventing the interaction between 2 m and MHC classI products renders infected cells unrecognizable by cytotoxic T cells.Therefore, as described in greater detail below in Example 4B,expression of the HCMV H301 gene product as a component of a therapeuticpalliative may be utilized in order to mitigate the host immune responseto viral NSP.

5. NONALPHAVIRUS PACKAGING SEQUENCE

Within another aspect of the invention, a packaging sequence derivedfrom a virus other than an alphavirus (for example, retrovirus,coronavirus, hepatitis B virus) is inserted into a tandem vector andpositioned between the first (inactivated) viral junction region and thesecond, modified viral junction region. Briefly, nonalphavirus packagingsequences signal the packaging of an RNA genome into a virus particlecorresponding to the source of the packaging sequence. For example, andas described in more detail below, a retroviral packaging sequence maybe utilized in order to package an alphavirus vector into a retroviralparticle using a retroviral packaging cell line. This is performed inorder to increase the efficiency of alphavirus vector transfer into analphavirus packaging cell line, or to alter the cell or tissue tropismof the alphavirus vector.

6. EXPRESSION OF MULTIPLE HETEROLOGOUS GENES

The genomic length and subgenomic length of mRNAs transcribed inwild-type alphavirus infected cells are polycistronic, coding for,respectively, the viral four non-structural proteins (NSPs) and fourstructural proteins (SPs). The genomic and subgenomic mRNAs aretranslated as polyproteins, and processing into the individualnonstructural and structural proteins is accomplished bypost-translational proteolytic cleavage, catalyzed by viral encoded NSP-and Sp- specific proteases, as well as cellular proteases.

In certain applications of the alphavirus vectors described herein, theexpression of more than one heterologous gene is desired. For example,in order to treat metabolic disorders such as Gaucher's syndrome,multiple administrations of alphavirus vectors or particles may berequired, since duration of the therapeutic palliative may be limited.Therefore, with certain embodiments of the invention it may be desirableto co-express in a target cell the Ad 2 E3 gene (see Example 4), alongwith a therapeutic palliative, such as the glucocerebrosidase gene (seeExample 17). In wild-type virus, however, the structural protein (“SP”)polycistronic message is translated into a single polyprotein which issubsequently processed into individual proteins by cleavage withSP-encoded proteases. Thus, expression of multiple heterologous genesfrom a polycistronic message requires a mechanism different from thewild-type virus, since the SP protease gene, or the peptides recognizedfor cleavage, are not present in the replacement region of thealphavirus vectors.

Therefore, within one embodiment of the invention alphavirus vectors maybe constructed by placing appropriate signals either ribosomereadthrough or internal ribosome entry between cistrons. One suchrepresentative method of expressing multiple heterologous genes is setforth below in Example 5.

In yet another embodiment of the invention, the placement of signalspromoting either ribosome readthrough or internal ribosome entryimmediately downstream of the disabled junction region vectorpKSSINBVdlJR is described (see Examples 3 and 5). In this vectorconfiguration, synthesis of subgenomic message cannot occur, however,the heterologous proteins are expressed from genomic length mRNA byeither ribosomal readthrough (scanning) or internal ribosome entry.Relative to wild-type, the low level of viral transcription with thisalphavirus vector would prolong the life of the infected target cell.

In still another embodiment of the invention, placement of signalspromoting either ribosome readthrough or internal ribosome entryimmediately downstream of the pKSSINBVdlJRsjr or pKSSINBV vectors isdescribed. Briefly, since synthesis of subgenomic mRNA occurs in cellsinfected with the pKSSINBVdlJRsjr and pKSSINBV vectors, placement ofeither a ribosome readthrough sequence or an internal ribosome entrysequence between the two heterologous genes permits translation of bothproteins encoded by the subgenomic mRNA polycistronic message. Further,additional heterologous genes can be placed in the subgenomic mRNAregion, provided that a suitable translation initiation signal residesat the 5′ end of the translational AUG start codon. The number ofheterologous gene(s) which can be inserted into the subgenomic mRNAregion, as described here, is limited only by the packaging constraintsof the vector.

Different sequences which allow either ribosome readthroughcap-independent translation, or internal ribosome entry may be placedinto Sindbis vectors pKSSINBVdlJR, pKSSINBV, pKSSINBVdlJRsjrc, orvectors encompassed by the eukaryotic layered vector initiation system,in the configurations as discussed above. The source of thesetranslation control sequences are the picornaviruses polio and EMCV, the5′ noncoding region of the human immunoglobulin heavy-chain bindingprotein, and a synthetic sequence of at least 15 bps corresponding inpart to the Kozak consensus sequence for efficient translationalinitiation. Although not described in detail here, these signals whichaffect translational initiation can also be placed downstream of thejunction region and between heterologous genes in all of the modifiedjunction region vectors described in Example 3.

As noted above, the alphavirus cDNA vector construct also includes a 3′sequence which controls transcription termination. A representativeexample of such a sequence is set forth in more detail below in Examples2 and 3.

7. TISSUE SPECIFIC EXPRESSION

Within other aspects of the present invention, alphavirus vectorconstructs are provided which are capable of expressing a desiredheterologous sequence only in a selected tissue. One such representativeexample is shown in FIG. 20. Briefly, as shown in FIG. 20A, arecombinant alphavirus vector is constructed such that upon introductionof the vector (FIG. 20A) into a target cell, internal inverted repeatsequences which flank the transcriptional control regions (e.g.,modified junction region) loop out (see FIG. 20B), thereby preventingviral transcription of subgenomic sequences (“G.O.I.”) from thesynthetic junction region.

On the other hand, activation of the vector can be attained if theinverted repeats are designed to also hybridize to a specific cellularRNA sequence which is characteristic of a selected tissue or cell type.Such cellular RNA disrupts the disabling stem loop structure, therebyallowing the formation of a more stable secondary stem loop structure(FIGS. 20C and 20D). This secondary stem loop structure allowstranscription of the subgenomic message by placing the junction regionback into its correct positional configuration.

Full-length alphavirus vectors can also be transcribed using thesecondary stem loop structure by taking advantage of the ability of theviral polymerase to switch templates during synthesis of the negativestrand using a strand hopping mechanism termed copy choice (King, RNAgenetics II, CRC Press, Inc., Boca Raton Fla., Domingo et al. (ed.), pp.150-185, 1988). Once a single successful round of transcription hasoccurred, the resulting RNA transcript does not contain inverted repeatsbecause they are deleted as a result of the polymerase copy choiceevent. This newly synthesized RNA molecule now functions as the primaryRNA vector transcript which will transcribe and express as any othernon-disabled genomic alphavirus vector previously described. In this RNAvector configuration, tissue or cell-specific activation of the disabledSindbis vector can be achieved if specific RNA sequences, present onlyin the targeted cell or tissue types, are used in the design of theinverted repeats. In this fashion alphaviruses such as Sindbis can beengineered to be tissue-specific expression vectors using similarinverted sequences described above.

Using this vector system to achieve tissue specific expression enables atherapeutic alphavirus vector or particle to be delivered systemicallyinto a patient. If the vector should infect a cell which does notexpress the appropriate RNA species, the vector will only be capable ofexpression nonstructural proteins and not the gene of interest.Eventually, the vector will be harmlessly degraded.

Use of the above-described vectors enables virtual tissue-specificexpression possible for a variety of therapeutic applications, includingfor example, targeting vectors for the treatment for various types ofcancers. This rationale relies on specific expression of tumor-specificmarkers such as the carcinoembryonic tumor specific antigen (CEA) andthe alpha-fetoprotein tumor marker. Briefly, utilizing suchtumor-specific RNA to target specific tumors allows for thetumor-specific expression of toxic molecules, lymphokines or pro-drugsdiscussed below. Such methods may be utilized for a wide variety oftumors, including for example, colorectal, lung, breast, ovary, bladderand prostate cancers because all these tumors express the CEA. Onerepresentative illustration of vectors suitable for use within thisaspect of the present invention is set forth in more detail below inExample 16.

Briefly, CEA was one of the first tumor-specific markers to bedescribed, along with the alpha-fetoprotein tumor marker. CEA is anormal glycoprotein in the embryonic tissue of the gut, pancreas andliver during the first two trimesters of fetal development (PathologicBasis of Disease, 3rd edition 1984, Robbins et al. (eds.)). Previously,CEA was believed to be specific for adenocarcinomas of the colon,however, with the subsequent development of more sensitiveradioimmunoassays it became apparent that CEA was presented in theplasma with many endodermally derived cancers, particularly pancreatic,gastric and broncogenic.

Within related aspects of the present invention, alphaviruscell-specific expression vectors may be constructed to express viralantigens, ribozyme, antisense sequences or immunostimulatory factorssuch as gamma-interferon (γ-IFN), IL-2 or IL-5 for the targetedtreatment of virus infected cell types. In particular, in order totarget alphavirus vectors to specific foreign organism orpathogen-infected cells, inverted repeats of the alphavirus vector maybe selected to hybridize to any pathogen-specific RNA, for instancetarget cells infected by pathogens such as HIV, CMV, HBV, HPV and HSV.

Within yet other aspects of the invention, specific organ tissues may betargeted for the treatment of tissue-specific metabolic diseasesutilizing gene replacement therapies. For example, the liver is animportant target tissue because it is responsible for many of the body'smetabolic functions and is associated with many metabolic geneticdisorders. Such diseases include many of the glycogen storage diseases,phenylketonuria, Gaucher's disease and familial hypercholesterolemia.Presently there are many liver-specific enzymes and markers which havebeen sequenced which may be used to engineer appropriate invertedrepeats for alphavirus vectors. Such liver-specific cDNAs includesequences encoding for S-adenosylmethione synthetase (Horikawa et al.,Biochem. Int. 25:81, 1991); lecithin; cholesterolacyl transferase (Rogneet al., Biochem. Biophys. Res. Commun. 148:161, 1987); as well as otherliver-specific cDNAs (Chin et al., Ann. N.Y. Acad. Sci. 478:120, 1986).Such a liver-specific alphavirus vector could be used to deliver the lowdensity lipoprotein receptor (Yamamoto et al., Cell 39:27, 1984) toliver cells for the treatment of familial hypercholesterolemia (Wilsonet al., Mol. Biol. Med. 7:223, 1990).

E. HETEROLOGOUS SEQUENCES

As noted above, a wide variety of nucleotide sequences may be carried bythe alphavirus vector constructs of the present invention. Preferably,the nucleotide sequences should be of a size sufficient to allowproduction of viable virus. Within the context of the present invention,the production of any measurable titer, for example, by plaque assay,luciferase assay, or β-galactosidase assay, of infectious virus onappropriate susceptible monolayers, is considered to be “production ofviable virus.” This may be, at a minimum, an alphavirus vector constructwhich does not contain any additional heterologous sequence. However,within other embodiments, the vector construct may contain additionalheterologous or foreign sequences. Within preferred embodiments, theheterologous sequence will comprise a heterologous sequence of at leastabout 100 bases, 2 kb, 3.5 kb, 5 kb, 7 kb, or even a heterologoussequence of at least about 8 kb.

As will be evident to one of ordinary skill in the art given thedisclosure provided herein, the efficiency of packaging and hence, viraltiter, is to some degree dependent upon the size of the sequence to bepackaged. Thus, in order to increase the efficiency of packaging and theproduction of viable virus, additional non-coding sequences may be addedto the vector construct. Moreover, within certain embodiments of theinvention it may be desired to increase or decrease viral titer. Thisincrease or decrease may be accomplished by increasing or decreasing thesize of the heterologous sequence, and hence the efficiency ofpackaging.

A wide variety of heterologous sequences may be included in the vectorconstruct, including for example sequences which encode palliatives suchas lymphokines, toxins, prodrugs, antigens which stimulate an immuneresponse, ribozymes, and proteins which assist or inhibit an immuneresponse, as well as antisense sequences (or sense sequences for“antisense applications”). As noted above, within various embodiments ofthe invention the alphavirus vector constructs provided herein maycontain (and express, within certain embodiments) two or moreheterologous sequences.

1. LYMPHOKINES

Within one embodiment of the invention, the heterologous sequenceencodes a lymphokine. Briefly, lymphokines act to proliferate, activate,or differentiate immune effectors cells. Representative examples oflymphokines include gamma interferon, tumor necrosis factor, IL-1, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13,IL-14, IL-15, GM-CSF, CSF-1 and G-CSF.

Within related embodiments of the invention, the heterologous sequenceencodes an immunomodulatory cofactor. Briefly, as utilized within thecontext of the present invention, “immunomodulatory cofactor” refers tofactors which, when manufactured by one or more of the cells involved inan immune response, or when added exogenously to the cells, causes theimmune response to be different in quality or potency from that whichwould have occurred in the absence of the cofactor. The quality orpotency of a response may be measured by a variety of assays known toone of skill in the art including, for example, in vitro assays whichmeasure cellular proliferation (e.g., ³H thymidine uptake), and in vitrocytotoxic assays (e.g., which measure ⁵¹Cr release) (see Warner et al.,AIDS Res. and Human Retroviruses 7:645-655, 1991).

Representative examples of immunomodulatory co-factors include alphainterferon (Finter et al., Drugs 42(5): 749-765, 1991; U.S. Pat. No.4,892,743; U.S. Pat. No. 4,966,843; WO 85/02862; Nagata et al., Nature284:316-320, 1980; Familletti et al., Methods in Enz. 78:387-394, 1981;Twu et al., Proc. Natl. Acad. Sci. USA 86:2046-2050, 1989; Faktor etal., Oncogene 5:867-872, 1990), beta interferon (Seif et al., J. Virol.65:664-671, 1991), gamma interferons (Radford et al., American Societyof Hepatology:2008-2015, 1991; Watanabe et al., PNAS 86:9456-9460, 1989;Gansbacher et al., Cancer Research 50:7820-7825, 1990; Maio et al., Can.Immunol. Immunother. 30:34-42, 1989; U.S. Pat. Nos. 4,762,791 and4,727,138), G-CSF (U.S. Pat. Nos. 4,999,291 and 4,810,643), GM-CSF (WO85/04188), TNFs (Jayaraman et al., J. Immunology 144:942-951, 1990),Interleukin-2 (IL-2) (Karupiah et al., J. Immunology 144:290-298, 1990;Weber et al., J. Exp. Med. 166:1716-1733, 1987; Gansbacher et al., J.Exp. Med. 172:1217-1224, 1990; U.S. Pat. No. 4,738,927), IL-4 (Tepper etal., Cell 57:503-512, 1989; Golumbek et al., Science 254:713-716, 1991;U.S. Pat. No. 5,017,691), IL-6 (Brakenhof et al., J. Immunol.139:4116-4121, 1987; WO 90/06370), IL-12, IL-15 (Grabstein et al.,Science 264:965-968, 1994; Genbank-EMBL Accession No. V03099), ICAM-1(Altman et al., Nature 338:512-514, 1989), ICAM-2, LFA-1, LFA-3, MHCclass I molecules, MHC class II molecules, ₂-microglobulin, chaperones,CD3, B7/BB1, MHC linked transporter proteins or analogues thereof.

The choice of which immunomodulatory cofactor to include within aalphavirus vector construct may be based upon known therapeutic effectsof the cofactor, or experimentally determined. For example, in chronichepatitis B infections alpha interferon has been found to be efficaciousin compensating a patient's immunological deficit and thereby assistingrecovery from the disease. Alternatively, a suitable immunomodulatorycofactor may be experimentally determined. Briefly, blood samples arefirst taken from patients with a hepatic disease. Peripheral bloodlymphocytes (PBLs) are restimulated in vitro with autologous orHLA-matched cells (e.g., EBV transformed cells), and transduced with analphavirus vector construct which directs the expression of animmunogenic portion of a hepatitis antigen and the immunomodulatorycofactor. Stimulated PBLs are used as effectors in a CTL assay with theHLA-matched transduced cells as targets. An increase in CTL responseover that seen in the same assay performed using HLA-matched stimulatorand target cells transduced with a vector encoding the antigen alone,indicates a useful immunomodulatory cofactor. Within one embodiment ofthe invention, the immunomodulatory cofactor gamma interferon isparticularly preferred.

Another example of an immunomodulatory cofactor is the B7/BB1costimulatory factor. Briefly, activation of the full functionalactivity of T cells requires two signals. One signal is provided byinteraction of the antigen-specific T cell receptor with peptides whichare bound to major histocompatibility complex (MHC) molecules, and thesecond signal, referred to as costimulation, is delivered to the T cellby antigen-presenting cells. Briefly, the second signal is required forinterleukin-2 (IL-2) production by T cells and appears to involveinteraction of the B7/BB1 molecule on antigen-presenting cells with CD28and CTLA-4 receptors on T lymphocytes (Linsley et al., J. Exp. Med.,173:721-730, 1991a, and J. Exp. Med., 174:561-570, 1991). Within oneembodiment of the invention, B7/BB1 may be introduced into tumor cellsin order to cause costimulation of CD8⁺ T cells, such that the CD8⁺ Tcells produce enough IL-2 to expand and become fully activated. TheseCD8⁺ T cells can kill tumor cells that are not expressing B7 becausecostimulation is no longer required for further CTL function. Vectorsthat express both the costimulatory B7/BB1 factor and, for example, animmunogenic HBV core protein, may be made utilizing methods which aredescribed herein. Cells transduced with these vectors will becomes moreeffective antigen-presenting cells. The HBV core-specific CTL responsewill be augmented from the fully activated CD8⁺ T cell via thecostimulatory ligand B7/BB1.

2. TOXINS

Within another embodiment of the invention, the heterologous sequenceencodes a toxin. Briefly, toxins act to directly inhibit the growth of acell. Representative examples of toxins include ricin (Lamb et al., Eur.J. Biochem. 148:265-270, 1985), abrin (Wood et al., Eur. J. Biochem.198:723-732, 1991; Evensen et al., J. of Biol. Chem. 266:6848-6852,1991; Collins et al., J. of Biol. Chem. 265:8665-8669, 1990; Chen etal., Fed. of Eur. Biochem Soc. 309:115-118, 1992), diphtheria toxin(Tweten et al., J. Biol. Chem. 260:10392-10394, 1985), cholera toxin(Mekalanos et al., Nature 306:551-557, 1983; Sanchez and Holmgren, PNAS86:481-485, 1989), gelonin (Stirpe et al., J. Biol. Chem. 255:6947-6953,1980), pokeweed (Irvin, Pharmac. Ther. 21:371-387, 1983), antiviralprotein (Barbieri et al., Biochem. J. 203:55-59, 1982; Irvin et al.,Arch. Biochem. & Biophys. 200:418-425, 1980; Irvin, Arch. Biochem. &Biophys. 169:522-528, 1975), tritin, Shigella toxin (Calderwood et al.,PNAS 84:4364-4368, 1987; Jackson et al., Microb. Path. 2:147-153, 1987),Pseudomonas exotoxin A (Carroll and Collier, J. Biol. Chem.262:8707-8711, 1987), herpes simplex virus thymidine kinase (HSVTK)(Field et al., J. Gen. Virol. 49:115-124, 1980), and E. coli. guaninephosphoribosyl transferase.

3. PRO-DRUGS

Within other embodiments of the invention, the heterologous sequenceencodes a “pro-drug”. Briefly, as utilized within the context of thepresent invention, “pro-drug” refers to a gene product that activates acompound with little or no cytotoxicity into a toxic product.Representative examples of such gene products include HSVTK and VZVTK(as well as analogues and derivatives thereof), which selectivelymonophosphorylate certain purine arabinosides and substituted pyrimidinecompounds, converting them to cytotoxic or cytostatic metabolites. Morespecifically, exposure of the drugs ganciclovir, acyclovir, or any oftheir analogues (e.g., FIAU, FIAC, DHPG) to HSVTK phosphorylates thedrug into its corresponding active nucleotide triphosphate form.

Representative examples of other pro-drugs which may be utilized withinthe context of the present invention include: E. coli guaninephosphoribosyl transferase which converts thioxanthine into toxicthioxanthine monophosphate (Besnard et al., Mol. Cell. Biol.7:4139-4141, 1987); alkaline phosphatase, which will convert inactivephosphorylated compounds such as mitomycin phosphate anddoxorubicin-phosphate to toxic dephosphorylated compounds; fungal (e.g.,Fusarium oxysporum) or bacterial cytosine deaminase, which will convert5-fluorocytosine to the toxic compound 5-fluorouracil (Mullen, PNAS89:33, 1992); carboxypeptidase G2, which will cleave the glutamic acidfrom para-N-bis (2-chloroethyl) aminobenzoyl glutamic acid, therebycreating a toxic benzoic acid mustard; and Penicillin-V amidase, whichwill convert phenoxyacetabide derivatives of doxorubicin and melphalanto toxic compounds (see generally, Vrudhula et al., J. of Med. Chem.36(7):919-923, 1993; Kern et al., Canc. Immun. Immunother.31(4):202-206, 1990).

4. ANTISENSE SEQUENCES

Within another embodiment of the invention, the heterologous sequence isan antisense sequence. Briefly, antisense sequences are designed to bindto RNA transcripts, and thereby prevent cellular synthesis of aparticular protein or prevent use of that RNA sequence by the cell.Representative examples of such sequences include antisense thymidinekinase, antisense dihydrofolate reductase (Maher and Dolnick, Arch.Biochem. & Biophys. 253:214-220, 1987; Bzik et al., PNAS 84:8360-8364,1987), antisense HER2 (Coussens et al., Science 230:1132-1139, 1985),antisense ABL (Fainstein et al., Oncogene 4:1477-1481, 1989), antisenseMyc (Stanton et al., Nature 310:423-425, 1984) and antisense ras, aswell as antisense sequences which block any of the enzymes in thenucleotide biosynthetic pathway. In addition, within other embodimentsof the invention antisense sequences to interferon and 2 microglobulinmay be utilized in order to decrease immune response.

In addition, within a further embodiment of the invention, antisense RNAmay be utilized as an anti-tumor agent in order to induce a potent ClassI restricted response. Briefly, in addition to binding RNA and therebypreventing translation of a specific mRNA, high levels of specificantisense sequences are believed to induce the increased expression ofinterferons (including gamma-interferon) due to the formation of largequantities of double-stranded RNA. The increased expression of gammainterferon, in turn, boosts the expression of MHC Class I antigens.Preferred antisense sequences for use in this regard include actin RNA,myosin RNA, and histone RNA. Antisense RNA which forms a mismatch withactin RNA is particularly preferred.

5. RIBOZYMES

Within other aspects of the present invention, alphavirus vectors areprovided which produce ribozymes upon infection of a host cell. Briefly,ribozymes are used to cleave specific RNAs and are designed such that itcan only affect one specific RNA sequence. Generally, the substratebinding sequence of a ribozyme is between 10 and 20 nucleotides long.The length of this sequence is sufficient to allow a hybridization withtarget RNA and disassociation of the ribozyme from the cleaved RNA.Representative examples for creating ribozymes include those describedin U.S. Pat. Nos. 5,116,742; 5,225,337 and 5,246,921. Particularlypreferred ribozymes for use within the present invention include thosedisclosed in more detail below in the Examples (e.g., Examples 18 and19).

6. PROTEINS AND OTHER CELLULAR CONSTITUENTS

Within other aspects of the present invention, a wide variety ofproteins or other cellular constituents may be carried by the alphavirusvector construct. Representative examples of such proteins includenative or altered cellular components, as well as foreign proteins orcellular constituents, found in for example, viruses, bacteria,parasites or fungus.

(a) Altered Cellular Components

Within one embodiment, alphavirus vector constructs are provided whichdirect the expression of an immunogenic, non-tumorigenic, alteredcellular component. As utilized herein, the term “immunogenic” refers toaltered cellular components which are capable, under the appropriateconditions, of causing an immune response. This response must becell-mediated, and may also include a humoral response. The term“non-tumorigenic” refers to altered cellular components which will notcause cellular transformation or induce tumor formation in nude mice.The phrase “altered cellular component” refers to proteins and othercellular constituents which are either associated with rendering a celltumorigenic, or are associated with tumorigenic cells in general, butare not required or essential for rendering the cell tumorigenic.

Before alteration, the cellular components may be essential to normalcell growth and regulation and include, for example, proteins whichregulate intracellular protein degradation, transcriptional regulation,cell-cycle control, and cell-cell interaction. After alteration, thecellular components no longer perform their regulatory functions and,hence, the cell may experience uncontrolled growth. Representativeexamples of altered cellular components include ras*, p53*, Rb*, alteredprotein encoded by the Wilms' tumor gene, ubiquitin*, mucin*, proteinencoded by the DCC, APC, and MCC genes, the breast cancer gene BRCA1*,as well as receptors or receptor-like structures such as neu, thyroidhormone receptor, platelet derived growth factor (PDGF) receptor,insulin receptor, epidermal growth factor (EGF) receptor, and the colonystimulating factor (CSF) receptor.

Within one embodiment of the present invention, alphavirus vectorconstructs are provided which direct the expression of anon-tumorigenic, altered ras (ras*) gene. Briefly, the ras* gene is anattractive target because it is causally linked to the neoplasticphenotype, and indeed may be necessary for the induction and maintenanceof tumorigenesis in a wide variety of distinct cancers, such aspancreatic carcinoma, colon carcinoma and lung andenocarcinoma. Inaddition, ras* genes are found in pre-neoplastic tumors and, therefore,immune intervention therapy may be applied prior to detection of amalignant tumor.

Normal ras genes are non-tumorigenic and ubiquitous in all mammals. Theyare highly conserved in evolution and appear to play an important rolein maintenance of the cell cycle and normal growth properties. Thenormal ras protein is a G-protein which binds GTP and has GTPaseactivity, and is involved in transmitting signals from the externalmilieu to the inside of the cell, thereby allowing a cell to respond toits environment. Ras* genes on the other hand alter the normal growthregulation of neoplastic cells by uncoupling cellular behavior from theenvironment, thus leading to the uncontrolled proliferation ofneoplastic cells. Mutation of the ras gene is believed to be an earlyevent in carcinogenesis (Kumar et al., Science 248:1101-1104, 1990)which, if treated early, may prevent tumorigenesis.

Ras* genes occur in a wide variety of cancers, including for example,pancreatic, colon, and lung adenocarcinomas. The spectrum of mutationsoccurring in the ras* genes found in a variety of cancers is quitelimited. These mutations alter the GTPase activity of the ras protein byconverting the normal on/off switch to a constitutive ON position.Tumorigenic mutations in ras* occur primarily (in vivo) in only 3codons: 12, 13 and 61. Codon 12 mutations are the most prevalent in bothhuman and animal tumors.

Table 1 below summarizes known in vivo mutations (codons 12, 13 and 61)which activate human ras, as well as potential mutations which have invitro transforming activity. Potential mutations in vitro transformingactivity were produced by the systematic substitution of amino acids forthe normal codon (e.g., other amino acids were substituted for thenormal glycine at position 12). In vitro mutations, while not presentlyknown to occur in humans or animals, may serve as the basis for ananti-cancer immunotherapeutic if they are eventually found to arise invivo.

TABLE 1 AMINO ACID SUBSTITUTIONS THAT ACTIVATE HUMAN RAS PROTEINS AminoGly Gly Ala Gln Glu Asn Lys Asp Acid Mutant 12 13 59 61 63 116 117 119Codon In vivo Val Asp Arg Arg Val His Asp Arg Leu Cys Ala Ser Phe Invitro Ala Ser Thr Val Lys His Glu His Asn Ala Ile Arg Glu Gln Cys AlaGlu Asn Asn His Ile Ile Met Leu Thr Lys Tyr Met Trp Phe Phe Ser Gly ThrTrp Tyr

Alterations as described above result in the he production of proteinscontaining novel coding sequence(s). The novel proteins encoded by thesesequence(s) may be used as a marker of tumorigenic cells, and an immuneresponse directed against these novel coding regions may be utilized todestroy tumorigenic cells containing the altered sequences (ras*).

Within another embodiment of the present invention, alphavirus vectorconstructs are provided which direct the expression of an altered p53(p53*) gene. Briefly, p53 is a nuclear phosphoprotein which wasoriginally discovered in extracts of transformed cells and thus wasinitially classified as an oncogene (Linzer and Levine, Cell 17:43-52,1979; Lane and Crawford, Nature 278:261-263, 1979). It was laterdiscovered that the original p53 cDNA clones were mutant forms of p53(Hinds et al., J. Virol. 63:739-746, 1989). It now appears that p53 is atumor suppressor gene which negatively regulates the cell cycle, andthat mutation of this gene may lead to tumor formation. Of coloncarcinomas that have been studied, 75%-80% show a loss of both p53alleles, one through deletion and the other through point mutation.Similar mutations are found in lung cancer, and in brain and breasttumors.

The majority of p53 mutations (e.g., p53*¹, p53*², etc.) are clusteredbetween amino acid residues 130 to 290 (see Levine et al., Nature351:453-456, 1991; see also the following references which describespecific mutations in more detail: Baker et al., Science 244:217-221,1989; Nigro et al., Nature 342:705-708, 1989 (p53 mutations cluster atfour “hot spots” which coincide with the four highly conserved regionsof the genes and these mutations are observed in human brain, breast,lung and colon tumors); Vogelstein, Nature 348:681-682, 1990; Takahashiet al., Science 246:491-494, 1989; Iggo et al., Lancet 335:675-679,1990; James et al., Proc. Natl. Acad. Sci. USA 86:2858-2862, 1989;Mackay et al., Lancet 11:1384-1385, 1988; Kelman et al., Blood74:2318-2324, 1989; Malkin et al., Science 250:1233-1238, 1990; Baker etal., Cancer Res. 50:7717-7722, 1991; Chiba et al., Oncogene 5:1603-1610,1990 (pathogenesis of early stage non-small cell lung cancer isassociated with somatic mutations in the p53 gene between codons 132 to283); Prosser et al., Oncogene 5:1573-1579, 1990 (mutations in the p53gene coding for amino acids 126 through 224 were identified in primarybreast cancer); Cheng and Hass, Mol. Cell. Biol. 10:5502-5509, 1990;Bartek et al., Oncogene 5:893-899, 1990; Rodrigues et al., Proc. Natl.Acad. Sci. USA 87:7555-7559, 1990; Menon et al., Proc. Natl. Acad. Sci.USA 87:5435-5439, 1990; Mulligan et al., Proc. Natl. Acad. Sci. USA87:5863-5867, 1990; and Romano et al., Oncogene 4:1483-1488, 1990(identification of a p53 mutation at codon 156 in human osteosarcomaderived cell line HOS-SL)).

Certain alterations of the p53 gene may be due to certain specifictoxins. For example, Bressac et al. (Nature 350:429-431, 1991) describesspecific G to T mutations in codon 249 in patients affected withhepatocellular carcinoma. One suggested causative agent of this mutationis aflatoxin B₁, a liver carcinogen which is known to be a foodcontaminant in Africa.

Four regions of the gene that are particularly affected occur atresidues 132-145, 171-179, 239-248, and 272-286. Three “hot spots” whichare found within these regions that are of particular interest occur atresidues 175, 248 and 273 (Levine et al., Nature 351:453-456, 1991).These alterations, as well as others which are described above, resultin the production of protein(s) which contain novel coding sequence(s).The novel proteins encoded by these sequences may be used as a marker oftumorigenic cells and an immune response directed against these novelcoding regions may be utilized to destroy tumorigenic cells containingthe altered sequence (p53*).

Once a sequence encoding the altered cellular component has beenobtained, it is necessary to ensure that the sequence encodes anon-tumorigenic protein. Various assays which assess the tumorigenicityof a particular cellular component are known and may easily beaccomplished. Representative assays include a rat fibroblast assay,tumor formation in nude mice or rats, colony formation in soft agar, andpreparation of transgenic animals, such as transgenic mice.

Tumor formation in nude mice or rats is a particularly important andsensitive method for determining the tumorigenicity of a particularcellular component. Nude mice lack a functional cellular immune system(i.e., do not possess CTLs), and therefore provide a useful in vivomodel in which to test the tumorigenic potential of cells. Normalnon-tumorigenic cells do not display uncontrolled growth properties ifinfected into nude mice. However, transformed cells will rapidlyproliferate and generate tumors in nude mice. Briefly, in one embodimentthe alphavirus vector construct is administered to syngeneic murinecells, followed by injection into nude mice. The mice are visuallyexamined for a period of 2 to 8 weeks after injection in order todetermine tumor growth. The mice may also be sacrificed and autopsied inorder to determine whether tumors are present. (Giovanella et al., J.Natl. Cancer Inst. 48:1531-1533, 1972; Furesz et al., Abnormal Cells,New Products and Risk, Hopps and Petricciani (eds.), Tissue CultureAssociation, 1985; and Levenbook et al., J. Biol. Std. 13:135-141,1985.)

Tumorigenicity may also be assessed by visualizing colony formation insoft agar (Macpherson and Montagnier, Vir. 23:291-294, 1964). Briefly,one property of normal non-tumorigenic cells is “contact inhibition”(i.e., cells will stop proliferating when they touch neighboring cells).If cells are plated in a semi-solid agar support medium, normal cellsrapidly become contact inhibited and stop proliferating, whereastumorigenic cells will continue to proliferate and form colonies in softagar.

Transgenic animals, such as transgenic mice, may also be utilized toassess the tumorigenicity of an altered cellular component. (Stewart etal., Cell 38:627-637, 1984; Quaife et al., Cell 48:1023-1034, 1987; andKoike et al., Proc. Natl. Acad. Sci. USA 86:5615-5619, 1989.) Intransgenic animals, the gene of interest may be expressed in all tissuesof the animal. This dysregulated expression of the transgene may serveas a model for the tumorigenic potential of the newly introduced gene.

If the altered cellular component is associated with making the celltumorigenic, then it is necessary to make the altered cellular componentnon-tumorigenic. For example, within one embodiment the sequence or geneof interest which encodes the altered cellular component is truncated inorder to render the gene product non-tumorigenic. The gene encoding thealtered cellular component may be truncated to a variety of sizes,although it is preferably to retain as much as possible of the alteredcellular component. In addition, it is necessary that any truncationleave intact at least some of the immunogenic sequences of the alteredcellular component. Alternatively, multiple translational terminationcodons may be introduced downstream of the immunogenic region. Insertionof termination codons will prematurely terminate protein expression,thus preventing expression of the transforming portion of the protein.

Within one embodiment, the ras* gene is truncated in order to render theras* protein non-tumorigenic. Briefly, the carboxy-terminal amino acidsof ras* functionally allow the protein to attach to the cell membrane.Truncation of these sequences renders the altered cellular componentnon-tumorigenic. Preferably, the ras* gene is truncated in the purinering binding site, for example around the sequence which encodes aminoacid number 110. The ras* gene sequence may be truncated such that aslittle as about 20 amino acids (including the altered amino acid(s)) areencoded by the alphavirus vector construct, although preferably, as manyamino acids as possible should be expressed (while maintainingnon-tumorigenicity).

Within another embodiment, the p53* protein is modified by truncation inorder to render the cellular component non-tumorigenic. As noted above,not all mutations of the p53 protein are tumorigenic, and therefore, notall mutations would have to be truncated. Nevertheless, within apreferred embodiment, p53* is truncated to a sequence which encodesamino acids 100 to 300, thereby including all four major “hot spots.”

Other altered cellular components which are oncogenic may also betruncated in order to render them non-tumorigenic. For example, both neuand bcr/abl may be truncated in order to render them non-tumorigenic.Non-tumorigenicity may be confirmed by assaying the truncated alteredcellular component as described above.

It should be noted, however, that if the altered cellular component isonly associated with non-tumorigenic cells in general, and is notrequired or essential for making the cell tumorigenic, then it is notnecessary to render the cellular component non-tumorigenic.Representative examples of such altered cellular components which arenot tumorigenic include Rb*, ubiquitin*, and mucin*.

As noted above, in order to generate an appropriate immune response, thealtered cellular component must also be immunogenic. Immunogenicity of aparticular sequence is often difficult to predict, although T cellepitopes often possess an immunogenic amphipathic alpha-helix component.In general, however, it is preferably to determine immunogenicity in anassay. Representative assays include an ELISA, which detects thepresence of antibodies against the newly introduced vector, as well asassays which test for T helper cells such as gamma-interferon assays,IL-2 production assays, and proliferation assays.

As noted above, within another aspect of the present invention, severaldifferent altered cellular components may be co-expressed in order toform a general anti-cancer therapeutic. Generally, it will be evident toone of ordinary skill in the art that a variety of combinations can bemade. Within preferred embodiments, this therapeutic may be targeted toa particular type of cancer. For example, nearly all colon cancerspossess mutations in ras, p53, DCC APC or MCC genes. An alphavirusvector construct which co-expresses a number of these altered cellularcomponents may be administered to a patient with colon cancer in orderto treat all possible mutations. This methodology may also be utilizedto treat other cancers. Thus, an alphavirus vector construct whichco-expresses mucin*, ras*, neu, BRCA1* and p53* may be utilized to treatbreast cancer.

(b) Antigens from foreign organisms or other pathogens

Within other aspects of the present invention, alphavirus vectorconstructs are provided which direct the expression of immunogenicportions of antigens from foreign organisms or other pathogens.Representative examples of such antigens include bacterial antigens(e.g., E. coli, streptococcal, staphylococcal, mycobacterial, etc.),fungal antigens, parasitic antigens, and viral antigens (e.g., influenzavirus, Human Immmunodeficiency Virus (“HIV”), Hepatitis A, B and C Virus(“HAV”, “HBV” and “HCV”, respectively), Human Papiloma Virus (“HPV”),Epstein-Barr Virus (“EBV”), Herpes Simplex Virus (“HSV”), Hantavirus,TTLV I, HTLV II and Cytomegalovirus (“CMV”). As utilized within thecontext of the present invention, “immunogenic portion” refers to aportion of the respective antigen which is capable, under theappropriate conditions, of causing an immune response (i.e.,cell-mediated or humoral). “Portions” may be of variable size, but arepreferably at least 9 amino acids long, and may include the entireantigen. Cell-mediated immune responses may be mediated through MajorHistocompatability Complex (“MHC”) class I presentation, MHC Class IIpresentation, or both.

Within one aspect of the invention, alphavirus vector constructs areprovided which direct the expression of immunogenic portions ofHepatitis B antigens. Briefly, the Hepatitis B genome is comprised ofcircular DNA of about 3.2 kilobases in length and has been wellcharacterized (Tiollais et al., Science 213:406-411, 1981; Tiollais etal., Nature 317:489-495, 1985; and Ganem and Varmus, Ann. Rev. Biochem.56:651-693, 1987; see also EP 0 278,940, EP 0 241,021, WO 88/10301, andU.S. Pat. Nos. 5,696,898 and 5,024,938, which are hereby incorporated byreference). The Hepatitis B virus presents several different antigens,including among others, three HB “S” antigens (HBsAgs), an HBc antigen(HBcAg), and HBe antigen (HBeAg), and an HBx antigen (HBxAg) (see Blumet al., TIG 5(5):154-158, 1989). Briefly, the HBeAg results fromproteolytic cleavage of a P22 pre-core intermediate and is secreted fromthe cell. HBeAg is found in serum as a 17 kD protein. The HBcAg is aprotein of 183 amino acids, and the HBxAg is a protein of 145 to 154amino acids, depending on subtype.

The HBsAgs (designated “large,” “middle” and “small”) are encoded bythree regions of the Hepatitis B genome: S, pre-S2 and pre-S1. The largeprotein, which has a length varying from 389 to 400 amino acids, isencoded by pre-S1, pre-S2, and S regions, and is found in glycosylatedand non-glycosylated forms. The middle protein is 281 amino acids longand is encoded by the pre-S2 and S regions. The small protein is 226amino acids long and is encoded by the S region. It exists in two forms,glycosylated (GP 27^(S)) and non-glycosylated (P24^(S)). If each ofthese regions are expressed separately, the pre-S1 region will code fora protein of approximately 119 amino acids, the pre-S2 region will codefor a protein of approximately 55 amino acids, and the S region willcode for a protein of approximately 226 amino acids.

As will be evident to one of ordinary skill in the art, variousimmunogenic portions of the above-described S antigens may be combinedin order to induce an immune response when administered by one of thealphavirus vector constructs described herein. In addition, due to thelarge immunological variability that is found in different geographicregions for the S open reading from of HBV, particular combinations ofantigens may be preferred for administration in particular geographicregions. Briefly, epitopes that are found in all human hepatitis B virusS samples are defined as determinant “a”. Mutually exclusive subtypedeterminants, however, have also been identified by two-dimensionaldouble immunodiffusion (Ouchterlony, Progr. Allergy 5:1, 1958). Thesedeterminants have been designated “d” or “y” and “w” or “r” (LeBouvier,J. Infect. 123:671, 1971; Bancroft et al., J. Immunol. 109:842, 1972;and Courouce et al., Bibl. Haematol. 42:1-158, 1976). The immunologicalvariability is due to single nucleotide substitutions in two areas ofthe hepatitis B virus S open reading frame, resulting in the followingamino acid changes: (1) exchange of lysine-122 to arginine in theHepatitis B virus S open reading frame causes a subtype shift from d toy, and (2) exchange of arginine-160 to lysine causes the shift fromsubtype r to w. In Africans, subtype ayw is predominant, whereas in theU.S. and northern Europe the subtype adw₂ is more abundant (MolecularBiology of the Hepatitis B Virus, McLachlan (ed.), CRC Press, 1991). Aswill be evident to one of ordinary skill in the art, it is generallypreferred to construct a vector for administration which is appropriateto the particular hepatitis B virus subtype which is prevalent in thegeographical region of administration. Subtypes of a particular regionmay be determined by two-dimensional double immunodiffusion or,preferably, by sequencing the S open reading frame of HBV virus isolatedfrom individuals within that region.

Also presented by HBV are pol (“HBV pol”), ORF 5, and ORF 6 antigens.Briefly, the polymerase open reading frame of HBV encodes reversetranscriptase activity found in virions and core-like particles ininfected livers. The polymerase protein consists of at least twodomains: the amino terminal domain which encodes the protein that primesreverse transcription, and the carboxyl terminal domain which encodesreverse transcriptase and RNase H activity. Immunogenic portions of HBVpol may be determined utilizing methods described herein (e.g., belowand in Example 13), utilizing alphavirus vector constructs describedbelow, and administered in order to generate an immune response within awarm-blooded animal. Similarly, other HBV antigens, such as ORF 5 andORF 6 (Miller et al., Hepatology 9:322-327, 1989) may be expressedutilizing alphavirus vector constructs as described herein.Representative examples of alphavirus vector constructs utilizing ORF 5and ORF 6 are set forth below in the examples.

As noted above, at least one immunogenic portion of a hepatitis Bantigen is incorporated into an alphavirus vector construct. Theimmunogenic portion(s) which are incorporated into the alphavirus vectorconstruct may be of varying length, although it is generally preferredthat the portions be at least 9 amino acids long and may include theentire antigen. Immunogenicity of a particular sequence is oftendifficult to predict, although T cell epitopes may be predictedutilizing computer algorithms such as TSITES (MedImmune, Maryland), inorder to scan coding regions for potential T-helper sites and CTL sites.From this analysis, peptides are synthesized and used as targets in anin vitro cytotoxic assay. Other assays, however, may also be utilized,including, for example, ELISA, which detects the presence of antibodiesagainst the newly introduced vector, as well as assays which test for Thelper cells, such as gamma-interferon assays, IL-2 production assaysand proliferation assays.

Immunogenic portions may also be selected by other methods. For example,the HLA A2.1 transgenic mouse has been shown to be useful as a model forhuman T-cell recognition of viral antigens. Briefly, in the influenzaand hepatitis B viral systems, the murine T cell receptor repertoirerecognizes the same antigenic determinants recognized by human T cells.In both systems, the CTL response generated in the HLA A2.1 transgenicmouse is directed toward virtually the same epitope as those recognizedby human CTLs of the HLA A2.1 haplotype (Vitiello et al., J. Exp. Med.173:1007-1015, 1991; Vitiello et al., Abstract of Molecular Biology ofHepatitis B Virus Symposia, 1992).

Particularly preferred immunogenic portions for incorporation intoalphavirus vector constructs include HBeAg, HBcAg and HBsAgs, asdescribed in greater detail below in Example 13.

Additional immunogenic portions of the hepatitis B virus may be obtainedby truncating the coding sequence at various locations including, forexample, the following sites: Bst UI, Ssp I, Ppu M1, and Msp I(Valenzuela et al., Nature 280:815-19, 1979; Valenzuela et al., AnimalVirus Genetics: ICN/UCLA Symp. Mol. Cell Biol., 1980, B. N. Fields andR. Jaenisch (eds.), pp. 57-70, New York: Academic). Further methods fordetermining suitable immunogenic portions as well as methods are alsodescribed below in the context of heptatitis C.

As noted above, more than one immunogenic portion may be incorporatedinto the alphavirus vector construct. For example, an alphavirus vectorconstruct may express (either separately or as one construct) all orimmunogenic portions of HBcAg, HBeAg, HBsAgs, HBxAg, as well asimmunogenic portions of HCV antigens.

7. SOURCES FOR HETEROLOGOUS SEQUENCES

Sequences which encode the above-described proteins may be readilyobtained from a variety of sources, including for example, depositoriessuch as the American Type Culture Collection (ATCC, Rockville, Md.), orfrom commercial sources such as British Bio-Technology Limited (Cowley,Oxford, England). Representative examples include BBG 12 (containing theGM-CSF gene coding for the mature protein of 127 amino acids); BBG 6(which contains sequences encoding gamma interferon), ATCC No. 39656(which contains sequences encoding TNF), ATCC No. 20663 (which containsequences encoding alpha interferon), ATCC Nos. 31902, 31902 and 39517(which contains sequences encoding beta interferon), ATCC No 67024(which contain a sequence which encodes Interleukin-1b); ATCC Nos.39405, 39452, 39516, 39626 and 39673 (which contains sequences encodingInterleukin-2); ATCC Nos. 59339, 59398, and 67326 (which containsequences encoding Interleukin-3); ATCC No. 57592 (which containssequences encoding Interleukin-4), ATCC Nos. 59394 and 57595 (whichcontains sequences encoding Interleukin-5), and ATCC No. 67153 (whichcontains sequences encoding Interleukin-6).

Sequences which encode altered cellular components as described abovemay be readily obtained from a variety of sources. For example, plasmidswhich contain sequences that encode altered cellular products may beobtained from a depository such as the American Type Culture Collection(ATCC, Rockville, Md.), or from commercial sources such as AdvancedBiotechnologies (Columbia, Md.). Representative examples of plasmidscontaining some of the above-described sequences include ATCC No. 41000(containing a G to T mutation in the 12th coding of ras), and ATCC No.41049 (containing a G to A mutation in the 12th codon).

Alternatively, plasmids which encode normal cellular components may alsobe obtained from depositories such as the ATCC (see, for example, ATCCNo. 41001, which contains a sequence which encodes the normal rasprotein; ATCC No. 57103, which encodes abl; and ATCC Nos. 59120 or59121, which encode the bcr locus) and mutated to form the alteredcellular component. Methods for mutagenizing particular sites mayreadily be accomplished using methods known in the art (see Sambrook etal., supra., 15.3 et seq.). In particular, point mutations of normalcellular components such as ras may readily be accomplished bysite-directed mutagenesis of the particular codon, for example, codons12, 13 or 61.

Sequences which encode the above-described viral antigens may likewisebe obtained from a variety of sources. For example, molecularly clonedgenomes which encode the hepatitis B virus may be obtained from sourcessuch as the American Type Culture Collection (ATCC, Rockville, Md.). Forexample, ATCC No. 45020 contains the total genomic DNA of hepatitis B(extracted from purified Dane particles) (see FIG. 3 of Blum et al., TIG5(5):154-158, 1989) in the Bam HI site of pBR322 (Moriarty et al., Proc.Natl. Acad. Sci. USA 78:2606-2610, 1981). Alternatively, cDNA sequenceswhich encode the above-mentioned heterologous sequences may be obtainedfrom cells which express or contain the sequences. Briefly, within oneembodiment, mRNA from a cell which expresses the gene of interest isreverse transcribed with reverse transcriptase using oligonucleotide dTor random primers. The single stranded cDNA may then be amplified by PCR(see U.S. Pat. Nos. 4,683,202; 4,683,195 and 4,800,159. See also PCRTechnology: Principles and Applications for DNA Amplification, Erlich(ed.), Stockton Press, 1989) utilizing oligonucleotide primerscomplementary to sequences on either side of desired sequences. Inparticular, a double-stranded DNA is denatured by heating in thepresence of heat stable Taq polymerase, sequence-specific DNA primers,dATP, dCTP, dGTP and dTTP. Double-stranded DNA is produced whensynthesis is complete. This cycle may be repeated many times, resultingin a factorial amplification of the desired DNA.

Sequences which encode the above-described proteins may also besynthesized, for example, on an Applied Biosystems Inc. DNA synthesizer(e.g., APB DNA synthesizer model 392 (Foster City, Calif.)).

F. EUKARYOTIC LAYERED VECTOR INITIATION SYSTEMS

Due to the size of a full-length genomic alphavirus cDNA clone, in vitrotranscription of full-length RNA molecules is rather inefficient. Thisresults in a lowered transfection efficiency, in terms of infectiouscenters of virus (as measured by plaque formation), relative to theamount of in vitro transcribed RNA transfected. Such inefficiency isalso relevant to the in vitro transcription of alphavirus expressionvectors. Testing of candidate cDNA clones and other alphavirus cDNAexpression vectors for their ability to initiate an infectious cycle orto direct the expression of a heterologous sequence would thus begreatly facilitated if a cDNA clone was transfected into susceptiblecells as a DNA molecule, which then directed the synthesis of viral RNAin vivo.

Therefore, within one aspect of the present invention DNA-based vectors(referred to as “Eukaryotic Layered Vector Initiation Systems”) areprovided which are capable of directing the synthesis of viral RNA invivo. In particular, eukaryotic layered vector initiation systems areprovided comprising a promoter which is capable of initiating the 5′synthesis of RNA from cDNA, a construct which is capable of autonomousreplication in a cell, the construct also being capable of expressing aheterologous nucleic acid sequence, and a 3′ sequence which controlstranscription termination. Briefly, such eukaryotic layered vectorinitiation systems provide a two-stage or “layered” mechanism whichcontrols expression of heterologous nucleotide sequences. The firstlayer initiates transcription of the second layer, and comprises apromoter which is capable of initiating the 5′ synthesis of RNA fromcDNA (e.g., a 5′ promoter), a 3′ transcription termination site, as wellas one or more splice sites and/or a polyadenylation site, if desired.Representative promoters suitable for use within the present inventioninclude both eukaryotic (e.g., pol I, II, or III) and prokaryoticpromoters, and inducible or non-inducible (i.e., constitutive)promoters, such as, for example, Murine Leukemia virus promoters (e.g.,MoMLV), metallothionein promoters, the glucocorticoid promoter,Drosophila actin 5C distal promoter, SV 40 promoter, heat shock protein65 promoter, heat shock protein 70 promoter, immunoglobulin promoters,Mouse polyoma virus promoter (“Py”), rous sarcoma virus (“RSV”), BKvirus and JC virus promoters, MMTV promoter, alphavirus junction region,CMV promoter, Adenovirous VA1RNA, rRNA promoter, tRNA methioninepromoter, CaMV 35S promoter, nopaline synthetase promoter, and the lacpromoter. The second layer comprises a vector construct which is capableof expressing one or more heterologous nucleotide sequences and ofreplication in a cell, either autonomously or in response to one or morefactors. Within one embodiment of the invention, the second layerconstruct may be an alphavirus vector construct as described above.

A wide variety of vector systems may be utilized as the first layer ofthe eukaryotic layered vector initiation system, including for example,viral vector constructs developed from DNA viruses such as thoseclassified in the Poxviridae, including for example canary pox virus orvaccinia virus (e.g., Fisher-Hoch et al., PNAS 86:317-321, 1989; Flexneret al., Ann. N.Y. Acad. Sci. 569:86-103, 1989; Flexner et al., Vaccine8:17-21, 1990; U.S. Pat. Nos. 4,603,112, 4,769,330 and 5,017,487; WO89/01973); Papoviridae such as BKV, JCV or SV40 (e.g., Mulligan et al.,Nature 277:108-114, 1979); Adenoviridae, such as adenovirus (e.g.,Berkner, Biotechniques 6:616-627, 1988; Rosenfeld et al., Science252:431-434, 1991); Parvoviridae, such as adeno-associated virus (e.g.,Samulski et al., J. Vir. 63:3822-3828, 1989; Mendelson et al., Virol166:154-165, 1988; PA 7/222,684); Herpesviridae, such as Herpes SimplexVirus (e.g., Kit, Adv. Exp. Med. Biol. 215:219-236, 1989); andHepadnaviridae (e.g., HBV), as well as certain RNA viruses whichreplicate through a DNA intermediate, such as the Retroviridae (see,e.g., U.S. Pat. No. 4,777,127, GB 2,200,651, EP 0,345,242 andWO91/02805; Retroviridae include leukemia in viruses such as MoMLV andimmunodeficiency viruses such as HIV, e.g., Poznansky, J. Virol.65:532-536, 1991).

Similarly, a wide variety of vector systems may be utilized as secondlayer of the eukaryotic layered vector initiation system, including forexample, vector systems derived from viruses of the following families:Picornaviridae (e.g., poliovirus, rhinovirus, coxsackieviruses),Caliciviridae, Togaviridae (e.g. alphavirus, rubella), Flaviviridae(e.g., yellow fever), Coronaviridae (e.g., HCV, TGEV, IBV, MHV, BCV),Bunyaviridae, Arenaviridae, Retroviridae (e.g., RSV, MoMLV, HIV, HTLV),hepatitis delta virus and Astrovirus. In addition, non-mammalian RNAviruses (as well as components derived therefrom) may also be utilized,including for example, bacterial and bacteriophage replicases, as wellas components derived from plant viruses, such as potexviruses (e.g.,PVX), carlaviruses (e.g., PVM), tobraviruses (e.g., TRV, PEBV, PRV),Tobamoviruses (e.g., TMV, ToMV, PPMV), luteoviruses (e.g.,PLRV),potyviruses (e.g., TEV, PPV, PVY), tombusviruses (e.g., CyRSV),nepoviruses (e.g., GFLV), bromoviruses (e.g., BMV), and topamoviruses.

The replication competency of the autocatalytic vector construct,contained within the second layer of the eukaryotic vector initiationsystem, may be measured by a variety of assays known to one of skill inthe art including, for example, ribonuclease protection assays whichmeasure increases in both positive-sense and negative-sense RNA overtime, in transfected cells, in the presence of an inhibitor of cellularRNA synthesis, such as dactinomycin, and assays which measure thesynthesis of a subgenomic RNA or expression of a heterologous reportergene in transfected cells.

Within particularly preferred embodiments of the invention, eukaryoticlayered vector initiation systems are provided that comprise a 5′promoter which is capable of initiating the synthesis of viral RNA fromcDNA, followed by a 5′ sequence which is capable of initiatingtranscription of an alphavirus, a nucleotide sequence encodingalphavirus nonstructural proteins, a viral junction region which iseither active or which has been inactivated such that viraltranscription of the subgenomic fragment is prevented, an alphavirus RNApolymerase recognition sequence, and a 3′ sequence which controlstranscription termination. Within various embodiments, the viraljunction region may be modified, such that viral transcription of thesubgenomic fragment is merely reduced, rather than inactivated. Withinother embodiments, a second viral junction region may be insertedfollowing the first inactivated viral junction region, the second viraljunction region being either active or modified such that viraltranscription of the subgenomic fragment is reduced.

Following transcription of an alphavirus cDNA vector construct, theresulting alphavirus RNA vector molecule is comprised of a 5′ sequencewhich is capable of initiating transcription of an alphavirus, anucleotide sequence encoding alphavirus nonstructural proteins, a viraljunction region, a heterologous nucleotide sequence, an alphavirus RNApolymerase recognition sequence, and a polyadenylate sequence.

Various aspects of the alphavirus cDNA vector constructs have beendiscussed above, including the 5′ sequence which is capable ofinitiating transcription of an alphavirus, the nucleotide sequenceencoding alphavirus nonstructural proteins, the viral junction region,including junction regions which have been inactivated such that viraltranscription of the subgenomic fragment is prevented, and thealphavirus RNA polymerase recognition sequence. In addition, modifiedjunction regions and tandem junction regions have also been discussedabove.

Within certain aspects of the present invention, methods are providedfor delivering a heterologous nucleotide sequence to a warm-bloodedanimal, comprising the step of administering a eukaryotic layered vectorinitiation system as described above, to a warm-blooded animal.Eukaryotic layered vector initiation systems may be administered towarm-blooded animals either directly (e.g., intravenously,intramuscularly, intraperitoneally, subcutaneously, orally, rectally,intraocularly, intranasally), or by various physical methods such aslipofection (Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417,1989), direct DNA injection (Acsadi et al., Nature 352:815-818, 1991);microprojectile bombardment (Williams et al., PNAS 88:2726-2730, 1991);liposomes of several types (see, e.g., Wang et al., PNAS 84:7851-7855,1987); CaPO₄ (Dubensky et al., PNAS 81:7529-7533, 1984); DNA ligand (Wuet al., J. of Biol. Chem. 264:16985-16987, 1989); administration ofnucleic acids alone (WO 90/11092); or administration of DNA linked tokilled adenovirus (Curiel et al., Hum. Gene Ther. 3:147-154, 1992); viapolycation compounds such as polylysine, utilizing receptor specificligands; as well as with psoralen inactivated viruses such as Sendai orAdenovirus. In addition, the eukaryotic layered vector initiationsystems may either be administered directly (i.e., in vivo), or to cellswhich have been removed (ex vivo), and subsequently returned.

Eukaryotic layered vector initiation systems may be administered to awarm-blooded animal for any of the therapeutic uses described herein,including for example, for the purpose of stimulating a specific immuneresponse; inhibiting the interaction of an agent with a host cellreceptor; to express a toxic palliative, including for example,conditional toxic palliatives; to immunologically regulate the immunesystem; to express markers, and for replacement gene therapy. These andother uses are discussed in more detail below.

In another embodiment of this aspect of the invention, eukaryoticlayered vector initiation systems can be utilized to direct theexpression of one or more recombinant proteins by eukaryotic cells. Asused herein, a “recombinant protein” refers to a protein, polypeptide,enzyme, or fragment thereof. Using this approach, proteins havingtherapeutic or other commercial application can be more cost-effectivelyproduced. Furthermore, proteins produced in eukaryotic cells may bepost-translationally modified (e.g., glycosylated, sulfated, acetylated,etc.), as compared to proteins produced in prokaryotic cells. Inaddition, such systems may be employed in the in vivo production ofvarious chemical compounds, e.g., fine or specialty chemicals.

Within this embodiment, a eukaryotic layered vector initiation systemencoding the desired protein, enzyme, or enzymatic pathway (as may berequired for the production of a desired chemical) is transformed,transfected, or otherwise introduced into a suitable eukaryotic cellline. Representative examples of proteins which can be produced usingsuch a system include, but are not limited to, insulin (see U.S. Pat.No. 4,431,740 and BE 885196A), hemoglobin (Lawn et al. Cell 21:647-51,1980), erythropoietin (EPO; see U.S. Pat. No. 4,703,008), megakaryocytegrowth and differentiation factor (MGDF), stem cell factor (SCF), G-CSF(Nagata et al. Nature 319:415-418, 1986), GM-CSF, M-CSF (see WO8706954), the flt3 ligand (Lyman, et al. (1993), Cell, vol. 75, pp.1157-1167), EGF, acidic and basic FGF, PDGF, members of the interleukinor interferon families, supra, neurotropic factors (e.g., BDNF;Rosenthal et al Endocrinology 129:1289-1294, 1991, NT-3; see WO 9103569,CNTF; see WO 9104316, NGF; see WO 9310150), coagulation factors (e.g.,factors VIII and IX), thrombolytic factors such as t-PA (see EP 292009,AU 8653302 and EP 174835) and streptokinase (see EP 407942), humangrowth hormone (see JP 94030582 and U.S. Pat. No. 4,745,069) and otheranimal somatotropins, and integrins and other cell adhesion molecules,such as ICAM-1 and ELAM. Genes encoding such recombinant proteins areamong the heterologous nucleic acid sequences of the invention. As thosein the art will appreciate, once characterized, any gene can readily becloned into a eukaryotic layered vector initiation system according tothe invention, followed by introduction into a suitable host cell andexpression of the desired gene.

In a preferred embodiment of this and other aspects of the invention,the eukaryotic layered vector initiation system is one derived from analphavirus vector, such as a Sindbis vector construct, which has beenadapted to replicate in one or more cell lines from a particulareukaryotic species, especially a mammalian species, such as humans. Forinstance, if the gene encoding the recombinant protein to be expressedis of human origin and the protein is intended for human therapeuticuse, production in a suitable human cell line may be preferred in orderthat the protein be post-translationally modified as would be expectedto occur in humans. This approach may be useful in further enhancingrecombinant protein production. Given the overall plasticity of analphaviral genome due to the infidelity of the viral replicase, variantstrains with an enhanced ability to establish high titer productiveinfection in selected eukaryotic cells (e.g., human, murine, canine,feline, etc.) can be isolated. Additionally, variant alphaviral strainshaving an enhanced ability to establish high titer persistent infectionin eukaryotic cells may also be isolated using this approach. Alphavirusexpression vectors can then be constructed from cDNA clones of thesevariant strains according to procedures provided herein.

Within another preferred embodiment of this aspect of the invention, theeukaryotic layered vector initiation system comprises a promoter forinitial alphaviral vector transcription that is transcriptionally activeonly in a differentiated cell type. It is well established thatalphaviral infection of cells in culture, in particular those derivedfrom hamster (e.g., baby hamster kidney cells) or chicken (e.g., chickenembryo fibroblasts), may result in cytoxicity. Thus, to produce a stablytransformed or transfected host cell line, the eukaryotic layered vectorinitiation system is preferably introduced into a host cell wherein thepromoter which enables the initial vector amplification is atranscriptionally inactive, but inducible, promoter. In a particularlypreferred embodiment, such as promoter is differentiation statedependent. In this configuration, activation of the promoter andsubsequent activation of the alphavirus DNA vector coincides withinduction of cell differentiation. Upon growth to a certain cell numberof such a stably transformed or transfected host cell line, theappropriate differentiation stimulus is provided, thereby initiatingtranscription of the vector construct and amplified expression of thedesired gene and encoded polypeptide(s). Many such differentiationstate-dependent promoters are known to those in the art, as are celllines which can be induced to differentiate by application of a specificstimulus. Representative examples include cell lines F9 and P19, HL60,and Freund erythroleukemic cell lines and HEL, which are activated byretinoic acid, horse serum, and DMSO, respectively.

G.ALPHAVIRUS PACKAGING CELL LINES

Within further embodiments of the invention, alphavirus packaging celllines are provided. In particular, within one aspect of the presentinvention, alphavirus packaging cell lines are provided wherein theviral structural proteins, supplied in trans from one or more stablyintegrated expression vectors, are able to encapsidate transfected,transduced, or intracellularly produced vector RNA transcripts in thecytoplasm and release infectious packaged vector particles through thecell membrane, thus creating an alphavirus vector producing cell line.Alphavirus RNA vector molecules, capable of replicating in the cytoplasmof the packaging cell, can be produced initially utilizing, for example,an SP6 RNA polymerase system to transcribe in vitro a cDNA vector cloneencoding the gene of interest and the alphavirus nonstructural proteins(described previously). Vector RNA transcripts are then transfected intothe alphavirus packaging cell line, such that the vector RNA replicatesto high levels, and is subsequently packaged by the viral structuralproteins, yielding infectious vector particles. Because of the extendedlength of the alphavirus cDNA molecule, the in vitro transcriptionprocess is inefficient. Further, only a fraction of the cells containedin a monolayer are typically transfected by most procedures.

In an effort to optimize vector producing cell line performance andtiter, two successive cycles of gene transfer may be performed. Inparticular, rather than directly transfecting alphavirus RNA vectormolecules into the final producing cell line, the vector may first betransfected into a primary alphavirus packaging cell line. Thetransfected primary packaging cell line releases infectious vectorparticles into the culture supernatants and these vector-containingsupernatants are subsequently used to transduce a fresh monolayer ofalphavirus packaging cells. Transduction into the final alphavirusvector producing cells is preferred over transfection because of itshigher RNA transfer efficiency into cells and optimized biologicalplacement of the vector in the cell. This leads to higher expression andhigher titer of packaged infectious recombinant alphavirus vector.

Within certain embodiments of the invention, alphavirus vector particlesmay fail to transduce the same packaging cell line because the cell lineproduces extracellular envelope proteins which block cellular receptorsfor alphavirus vector particle attachment, a second type of alphavirusvector particle is generated which maintains the ability to transducethe alphavirus packaging cells. This second type of viral particle isproduced by a packaging cell line known as a “hopping cell line,” whichproduces transient vector particles as the result of being transfectedwith in vitro transcribed alphavirus RNA vector transcripts. Briefly,the hopping cell line is engineered to redirect the receptor tropism ofthe transiently produced vector particles by providing alternative viralenvelope proteins which redirect alphavirus vectors to differentcellular receptors, in a process termed pseudotyping. Two primaryapproaches have been devised for alphavirus vector particlepseudotyping. The first approach consists of an alphavirus packagingcell line expressing the vesicular stomatitis virus G protein (VSV-G).The second approach for producing a pseudotyped alphavirus vectorparticle is to use currently available retroviral packaging cell linescontaining retroviral gag/pol and env sequences which would be capableof packaging an alphavirus RNA vector containing a retroviral packagingsequence (e.g., WO 92/05266).

Within other embodiments of the invention, a second approach has alsobeen devised in which a stably integrated DNA expression vector is usedto produce the alphavirus vector RNA molecule, which, as in the firstapproach, maintains the autocatalytic ability to self-replicate. Thisapproach allows for continued vector expression over extended periods ofculturing because the integrated DNA vector expression system ismaintained through a drug selection marker and the DNA system willconstitutively express unaltered RNA vectors which cannot be diluted outby defective RNA copies. In this “alphavirus producer cell line”configuration, the DNA-based alphavirus vector is introduced initiallyinto the packaging cell line by transfection, since size restrictionscould prevent packaging of the expression vector into a viral vectorparticle for transduction. Also, for this configuration, the SP6 RNApolymerase recognition site of the plasmid, previously used totranscribe vector RNA in vitro, is replaced with another appropriatepromoter sequence defined by the parent cell line used. In addition,this plasmid sequence also contains a selection marker different fromthat used to create the packaging cell line.

The expression of alphavirus proteins and/or vector RNA above certainlevels may result in cytotoxic effects in packaging cell lines.Therefore, within certain embodiments of the invention, it may bedesirable for these elements to be expressed only after thepackaging/producer cells have been propagated to a certain criticaldensity. For this purpose, additional packaging or producer cell linemodifications are made whereby the structural proteins necessary forpackaging are synthesized only after induction by the RNA vector itselfor some other stimulus. Also, other modifications allow for theindividual expression of these proteins under the control of separateinducible elements, by utilizing expression vectors which unlink thegenes encoding these proteins. In addition, expression of the integratedvector molecule itself, in some instances, is controlled by yet anotherinducible system. This configuration results in a cascade of eventsfollowing induction, that ultimately leads to the production of packagedvector particles.

H. METHODS FOR UTILIZING ALPHAVIRUS VECTORS

1. Immunostimulation

Within other aspects of the present invention, compositions and methodsare provided for administering an alphavirus vector construct which iscapable of preventing, inhibiting, stabilizing or reversing infectious,cancerous, auto-immune or immune diseases. Representative examples ofsuch diseases include viral infections such as HIV, HBV HTLV I, HTLV II,CMV, EBV and HPV, melanomas, diabetes, graft vs. host disease,Alzheimer's disease and heart disease.

More specifically, within one aspect of the present invention,compositions and methods are provided for stimulating an immune response(either humoral or cell-mediated) to a pathogenic agent, such that thepathogenic agent is either killed or inhibited. Representative examplesof pathogenic agents include bacteria, fungi, parasites, viruses andcancer cells.

Within one embodiment of the invention the pathogenic agent is a virus,and methods are provided for stimulating a specific immune response andinhibiting viral spread by using recombinant alphavirus viral particlesdesigned to deliver a vector construct that directs the expression of anantigen or modified form thereof to susceptible target cells capable ofeither (1) initiating an immune response to the viral antigen or (2)preventing the viral spread by occupying cellular receptors required forviral interactions. Expression of the vector nucleic acid encodedprotein may be transient or stable with time. Where an immune responseis to be stimulated to a pathogenic antigen, the recombinant alphavirusis preferably designed to express a modified form of the antigen whichwill stimulate an immune response and which has reduced pathogenicityrelative to the native antigen. This immune response is achieved whencells present antigens in the correct manner, i.e., in the context ofthe MHC class I and/or II molecules along with accessory molecules suchas CD3, ICAM-1, ICAM-2, LFA-1, or analogues thereof (e.g., Altmann etal., Nature 338:512, 1989). Cells infected with alphavirus vectors areexpected to do this efficiently because they closely mimic genuine viralinfection and because they: (a) are able to infect non-replicatingcells, (b) do not integrate into the host cell genome, (c) are notassociated with any life threatening diseases, and (d) express highlevels of heterologous protein. Because of these differences, alphavirusvectors can easily be thought of as safe viral vectors which can be usedon healthy individuals for vaccine use.

This aspect of the invention has a further advantage over other systemsthat might be expected to function in a similar manner, in that thepresenter cells are fully viable and healthy, and low levels of viralantigens, relative to heterologous genes, are expressed. This presents adistinct advantage since the antigenic epitopes expressed can be alteredby selective cloning of sub-fragments of the gene for the antigen intothe recombinant alphavirus, leading to responses against immunogenicepitopes which may otherwise be overshadowed by immunodominant epitopes.Such an approach may be extended to the expression of a peptide havingmultiple epitopes, one or more of the epitopes being derived fromdifferent proteins. Further, this aspect of the invention allowsefficient stimulation of cytotoxic T lymphocytes (CTL) directed againstantigenic epitopes, and peptide fragments of antigens encoded bysub-fragments of genes, through intracellular synthesis and associationof these peptide fragments with MHC Class I molecules. This approach maybe utilized to map major immunodominant epitopes for CTL induction.

An immune response may also be achieved by transferring to anappropriate immune cell (such as T lymphocyte) the gene for the specificT cell receptor which recognizes the antigen of interest (in the contextof an appropriate MHC molecule if necessary), for an immunoglobulinwhich recognizes the antigen of interest, or for a hybrid of the twowhich provides a CTL response in the absence of the MHC context. Thus,the recombinant alphavirus infected cells may be used as animmunostimulant, immunomodulator, or vaccine.

In another embodiment of the invention, methods are provided forproducing inhibitor palliatives wherein alphavirus vectors deliver andexpress defective interfering viral structural proteins, which inhibitviral assembly. Such vectors may encode defective gag, pol, env or otherviral particle proteins or peptides and these would inhibit in adominant fashion the assembly of viral particles. This occurs becausethe interaction of normal subunits of the viral particle is disturbed byinteraction with the defective subunits.

In another embodiment of the invention, methods are provided for theexpression of inhibiting peptides or proteins specific for viralprotease. Briefly, viral protease cleaves the viral gag and gag/polproteins into a number of smaller peptides. Failure of this cleavage inall cases leads to complete inhibition of production of infectiousretroviral particles. As an example, the HIV protease is known to be anaspartyl protease and these are known to be inhibited by peptides madefrom amino acids from protein or analogues. Vectors to inhibit HIV willexpress one or multiple fused copies of such peptide inhibitors.

Another embodiment involves the delivery of suppressor genes which, whendeleted, mutated, or not expressed in a cell type, lead to tumorigenesisin that cell type. Reintroduction of the deleted gene by means of aviral vector leads to regression of the tumor phenotype in these cells.Examples of such cancers are retinoblastoma and Wilms Tumor. Sincemalignancy can be considered to be an inhibition of cellular terminaldifferentiation compared with cell growth, the alphavirus vectordelivery and expression of gene products which lead to differentiationof a tumor should also, in general, lead to regression.

In yet another embodiment, the alphavirus vector provides a therapeuticeffect by transcribing a ribozyme (an RNA enzyme) (Haseloff and Gerlach,Nature 334:585, 1989) which will cleave and hence inactivate RNAmolecules corresponding to a pathogenic function. Since ribozymesfunction by recognizing a specific sequence in the target RNA and thissequence is normally 12 to 17 bp, this allows specific recognition of aparticular RNA species such as a RNA or a retroviral genome. Additionalspecificity may be achieved in some cases by making this a conditionaltoxic palliative (see below).

One way of increasing the effectiveness of inhibitory palliatives is toexpress viral inhibitory genes in conjunction with the expression ofgenes which increase the probability of infection of the resistant cellby the virus in question. The result is a nonproductive “dead-end” eventwhich would compete for productive infection events. In the specificcase of HIV, vectors may be delivered which inhibit HIV replication (byexpressing anti-sense tat, etc., as described above) and alsooverexpress proteins required for infection, such as CD4. In this way, arelatively small number of vector-infected HIV-resistant cells act as a“sink” or “magnet” for multiple nonproductive fusion events with freevirus or virally infected cells.

2. Blocking Agents

Many infectious diseases, cancers, autoimmune diseases, and otherdiseases involve the interaction of viral particles with cells, cellswith cells, or cells with factors. In viral infections, viruses commonlyenter cells via receptors on the surface of susceptible cells. Incancers, cells may respond inappropriately or not at all to signals fromother cells or factors. In autoimmune disease, there is inappropriaterecognition of “self” markers. Within the present invention, suchinteractions may be blocked by producing, in vivo, an analogue to eitherof the partners in an interaction.

This blocking action may occur intracellularly, on the cell membrane, orextracellularly. The blocking action of a viral or, in particular, analphavirus vector carrying a gene for a blocking agent, can be mediatedeither from inside a susceptible cell or by secreting a version of theblocking protein to locally block the pathogenic interaction.

In the case of HIV, the two agents of interaction are the gp 120/gp 41envelope protein and the CD4 receptor molecule. Thus, an appropriateblocker would be a vector construct expressing either an HIV envanalogue that blocks HIV entry without causing pathogenic effects, or aCD4 receptor analogue. The CD4 analogue would be secreted and wouldfunction to protect neighboring cells, while the gp 120/gp 41 issecreted or produced only intracellularly so as to protect only thevector-containing cell. It may be advantageous to add humanimmunoglobulin heavy chains or other components to CD4 in order toenhance stability or complement lysis. Delivery of an alphavirus vectorencoding such a hybrid-soluble CD4 to a host results in a continuoussupply of a stable hybrid molecule. Efficacy of treatment can be assayedby measuring the usual indicators of disease progression, includingantibody level, viral antigen production, infectious HIV levels, orlevels of nonspecific infections.

3. Expression of Palliatives

Techniques similar to those described above can be used to producerecombinant alphavirus vector constructs which direct the expression ofan agent (or “palliative”) which is capable of inhibiting a function ofa pathogenic agent or gene. Within the present invention, “capable ofinhibiting a function” means that the palliative either directlyinhibits the function or indirectly does so, for example, by convertingan agent present in the cells from one which would not normally inhibita function of the pathogenic agent to one which does. Examples of suchfunctions for viral disease include adsorption, replication, geneexpression, assembly, and exit of the virus from infected cells.Examples of such functions for a cancerous cell or cancer-promotinggrowth factor include viability, cell replication, alteredsusceptibility to external signals (e.g., contact inhibition), and lackof production or production of mutated forms of anti-oncogene proteins.

(a) Inhibitor Palliatives

In one aspect of the present invention, the alphavirus vector constructdirects the expression of a gene which can interfere with a function ofa pathogenic agent, for instance in viral or malignant diseases. Suchexpression may either be essentially continuous or in response to thepresence in the cell of another agent associated either with thepathogenic condition or with a specific cell type (an “identifyingagent”). In addition, vector delivery may be controlled by targetingvector entry specifically to the desired cell type (for instance, avirally infected or malignant cell) as discussed above.

One method of administration is leukophoresis, in which about 20% of anindividual's PLBs are removed at any one time and manipulated in vitro.Thus, approximately 2×10⁹ cells may be treated and replaced. Repeattreatments may also be performed. Alternatively, bone marrow may betreated and allowed to amplify the effect as described above. Inaddition, packaging cell lines producing a vector may be directlyinjected into a subject, allowing continuous production of recombinantvirions.

In one embodiment, alphavirus vectors which express RNA complementary tokey pathogenic gene transcripts (for example, a viral gene product or anactivated cellular oncogene) can be used to inhibit translation of thattranscript into protein, such as the inhibition of translation of theHIV tat protein. Since expression of this protein is essential for viralreplication, cells containing the vector would be resistant to HIVreplication.

In a second embodiment, where the pathogenic agent is a single-strandedvirus having a packaging signal, RNA complementary to the viralpackaging signal (e.g., an HIV packaging signal when the palliative isdirected against HIV) is expressed, so that the association of thesemolecules with the viral packaging signal will, in the case ofretroviruses, inhibit stem loop formation or tRNA primer bindingrequired for proper encapsidation or replication of the alphavirus RNAgenome.

In a third embodiment, an alphavirus vector may be introduced whichexpresses a palliative capable of selectively inhibiting the expressionof a pathogenic gene, or a palliative capable of inhibiting the activityof a protein produced by the pathogenic agent. In the case of HIV, oneexample is a mutant tat protein which lacks the ability to transactivateexpression from the HIV LTR and interferes (in a transdominant manner)with the normal functioning of tat protein. Such a mutant has beenidentified for HTLV II tat protein (“XII Leu⁵” mutant; see Wachsman etal., Science 235:674, 1987). A mutant transrepressor tat should inhibitreplication much as has been shown for an analogous mutant repressor inHSV-1 (Friedmann et al., Nature 335:452, 1988).

Such a transcriptional repressor protein may be selected for in tissueculture using any viral-specific transcriptional promoter whoseexpression is stimulated by a virus-specific transactivating protein (asdescribed above). In the specific case of HIV, a cell line expressingHIV tat protein and the HSVTK gene driven by the HIV promoter will diein the presence of ACV. However, if a series of mutated tat genes areintroduced to the system, a mutant with the appropriate properties(i.e., represses transcription from the HIV promoter in the presence ofwild-type tat) will grow and be selected. The mutant gene can then bereisolated from these cells. A cell line containing multiple copies ofthe conditionally lethal vector/tat system may be used to assure thatsurviving cell clones are not caused by endogenous mutations in thesegenes. A battery of randomly mutagenized tat genes are then introducedinto these cells using a “rescuable” alphavirus vector (i.e., one thatexpresses the mutant tat protein and contains a bacterial origin ofreplication and drug resistance marker for growth and selection inbacteria). This allows a large number of random mutations to beevaluated and permits facile subsequent molecular cloning of the desiredmutant cell line. This procedure may be used to identify and utilizemutations in a variety of viral transcriptional activator/viral promotersystems for potential antiviral therapies.

4. Conditional Toxic Palliatives

Another approach for inhibiting a pathogenic agent is to express apalliative which is toxic for the cell expressing the pathogeniccondition. In this case, expression of the palliative from the vectorshould be limited by the presence of an entity associated with thepathogenic agent, such as a specific viral RNA sequence identifying thepathogenic state, in order to avoid destruction of nonpathogenic cells.

In one embodiment of this method, a recombinant alphavirus vectorcarries a vector construct containing a toxic gene (as discussed above)expressed from a cell-specific responsive vector. In this manner,rapidly replicating cells, which contain the RNA sequences capable ofactivating the cell-specific responsive vectors, are preferentiallydestroyed by the cytotoxic agent produced by the alphavirus vectorconstruct.

In a similar manner to the preceding embodiment, the alphavirus vectorconstruct can carry a gene for phosphorylation, phosphoribosylation,ribosylation, or other metabolism of a purine- or pyrimidine-based drug.This gene may have no equivalent in mammalian cells and might come fromorganisms such as a virus, bacterium, fungus, or protozoan. An exampleof this would be the E. coli guanine phosphoribosyl transferase geneproduct, which is lethal in the presence of thioxanthine (see Besnard etal., Mol. Cell. Biol. 7:4139-4141, 1987). Conditionally lethal geneproducts of this type (also referred to as “pro-drugs” above) haveapplication to many presently known purine- or pyrimidine-basedanticancer drugs, which often require intracellular ribosylation orphosphorylation in order to become effective cytotoxic agents. Theconditionally lethal gene product could also metabolize a nontoxic drugwhich is not a purine or pyrimidine analogue to a cytotoxic form (seeSearle et al., Brit. J. Cancer 53:377-384, 1986).

Mammalian viruses in general tend to have “immediate early” genes whichare necessary for subsequent transcriptional activation from other viralpromoter elements. RNA sequences of this nature are excellent candidatesfor activating alphavirus vectors intracellular signals (or “identifyingagents”) of viral infection. Thus, conditionally lethal genes expressedfrom alphavirus cell-specific vectors responsive to these viral“immediate early” gene products could specifically kill cells infectedwith any particular virus. Additionally, since the human and interferonpromoter elements are transcriptionally activated in response toinfection by a wide variety of nonrelated viruses, the introduction ofvectors expressing a conditionally lethal gene product like HSVTK, forexample, in response to interferon production could result in thedestruction of cells infected with a variety of different viruses.

In another aspect of the present invention, the recombinant alphavirusviral vector carries a vector construct that directs the expression of agene product capable of activating an otherwise inactive precursor intoan active inhibitor of the pathogenic agent. For example, the HSVTK geneproduct may be used to more effectively metabolize potentially antiviralnucleoside analogues such as AZT or ddC. The HSVTK gene may be expressedunder the control of a cell-specific responsive vector and introducedinto these cell types. AZT (and other nucleoside antivirals) must bemetabolized by cellular mechanisms to the nucleotide triphosphate formin order to specifically inhibit retroviral reverse transcriptase, andthus, HIV replication (Furmam et al., Proc. Natl. Acad. Sci. USA83:8333-8337, 1986). Constitutive expression of HSVTK (a nucleoside andnucleoside kinase with very broad substrate specificity) results in moreeffective metabolism of these drugs to their biologically activenucleotide triphosphate form. AZT or ddC therapy will thereby be moreeffective, allowing lower doses, less generalized toxicity, and higherpotency against productive infection. Additional nucleoside analogueswhose nucleotide triphosphate forms show selectivity for retroviralreverse transcriptase but, as a result of the substrate specificity ofcellular nucleoside and nucleotide kinases are not phosphorylated, willbe made more efficacious.

Administration of these alphavirus vectors to human T cell andmacrophage/monocyte cell lines can increase their resistance to HIV inthe presence of AZT and ddC compared to the same cells withoutretroviral vector treatment. Treatment with AZT would be at lower thannormal levels to avoid toxic side effects but still efficiently inhibitthe spread of HIV. The course of treatment would be as described for theblocker.

In one embodiment, the recombinant alphavirus vector carries a genespecifying a product which is not in itself toxic but, when processed ormodified by a protein such as a protease specific to a viral or otherpathogen, is converted into a toxic form. For example, the recombinantalphavirus could carry a gene encoding a proprotein for ricin A chain,which becomes toxic upon processing by the HIV protease. Morespecifically, a synthetic inactive proprotein form of the toxin ricin ordiphtheria A chains could be cleaved to the active form by arranging forthe HIV virally encoded protease to recognize and cleave off anappropriate “pro” element.

In another embodiment, the alphavirus construct may express a “reportingproduct” on the surface of the target cells in response to the presenceof an identifying agent in the cells (such as expression of a viralgene). This surface protein can be recognized by a cytotoxic agent, suchas antibodies for the reporting protein, or by cytotoxic T cells. In asimilar manner, such a system can be used as a detection system (seebelow) to simply identify those cells having a particular gene whichexpresses an identifying protein.

Similarly, in another embodiment, a surface protein could be expressedwhich would itself be therapeutically beneficial. In the particular caseof HIV, expression of the human CD4 protein specifically in HIV-infectedcells may be beneficial in two ways:

1. Binding of CD4 to HIV env intracellularly could inhibit the formationof viable viral particles, much as soluble CD4 has been shown to do forfree virus, but without the problem of systematic clearance and possibleimmunogenicity, since the protein will remain membrane bound and isstructurally identical to endogenous CD4 (to which the patient should beimmunologically tolerant).

2. Since the CD4/HIV env complex has been implicated as a cause of celldeath, additional expression of CD4 (in the presence of excess HIV-envpresent in HIV-infected cells) leads to more rapid cell death and thusinhibits viral dissemination. This may be particularly applicable tomonocytes and macrophages, which act as a reservoir for virus productionas a result of their relative refractility to HIV-induced cytotoxicity(which, in turn, is apparently due to the relative lack of CD4 on theircell surfaces).

In another embodiment, the alphavirus vector codes for a ribozyme whichwill cleave and inactivate RNA molecules essential for viability of thevector infected cell. By making ribozyme production dependent on aspecific RNA sequence corresponding to the pathogenic state, such as HIVtat, toxicity is specific to the pathogenic state.

5. Expression of Markers

The above-described technique of expressing a palliative in a cell inresponse to a specific RNA sequence can also be modified to enabledetection of a particular gene in a cell which expresses an identifyingprotein (for example, a gene carried by a particular virus), and henceenable detection of cells carrying that virus. In addition, thistechnique enables the detection of viruses (such as HIV) in a clinicalsample of cells carrying an identifying protein associated with thevirus.

This modification can be accomplished by providing a genome coding for aproduct, the presence of which can be readily identified (the “markerproduct”), in an alphavirus vector which responds to the presence of theidentifying protein in the infected cells. For example, HIV, when itinfects suitable cells, makes tat and rev. The indicator cells can thusbe provided with a genome (such as by infection with an appropriaterecombinant alphavirus) which codes for a marker gene, such as thealkaline phosphatase gene, β-galactosidase gene, or the luciferase genewhich is expressed by the recombinant alphavirus upon activation by thetat and/or rev RNA transcript. In the case of β-galactosidase oralkaline phosphatase, exposing the cells to substrate analogues resultsin a color or fluorescence change if the sample is positive for HIV. Inthe case of luciferase, exposing the sample to luciferin will result inluminescence if the sample is positive for HIV. For intracellularenzymes such as β-galactosidase, the viral titre can be measureddirectly by counting colored or fluorescent cells, or by making cellextracts and performing a suitable assay. For the membrane bond form ofalkaline phosphatase, virus titre can also be measured by performingenzyme assays on the cell surface using a fluorescent substrate. Forsecreted enzymes, such as an engineered form of alkaline phosphatase,small samples of culture supernatant are assayed for activity, allowingcontinuous monitoring of a single culture over time. Thus, differentforms of this marker system can be used for different purposes. Theseinclude counting active virus, or sensitively and simply measuring viralspread in a culture and the inhibition of this spread by various drugs.

Further specificity can be incorporated into the preceding system bytesting for the presence of the virus either with or withoutneutralizing antibodies to that virus. For example, in one portion ofthe clinical sample being tested, neutralizing antibodies to HIV may bepresent; whereas in another portion there would be no neutralizingantibodies. If the tests were negative in the system where there wereantibodies and positive where there were no antibodies, this wouldassist in confirming the presence of HIV.

Within an analogous system for an in vitro assay, the presence of aparticular gene, such as a viral gene, may be determined in a cellsample. In this case, the cells of the sample are infected with asuitable alphavirus vector which carries the reporter gene which is onlyexpressed in the presence of the appropriate viral RNA transcript. Thereporter gene, after entering the sample cells, will express itsreporting product (such as β-galactosidase or luciferase) only if thehost cell expresses the appropriate viral proteins.

These assays are more rapid and sensitive, since the reporter gene canexpress a greater amount of reporting product than identifying agentpresent, which results in an amplification effect.

6. Immune Down-Regulation

As briefly described above, the present invention also providesrecombinant alphavirus which carry a vector construct capable ofsuppressing one or more elements of the immune system in target cellsinfected with the alphavirus.

Briefly, specific down-regulation of inappropriate or unwanted immuneresponses, such as in chronic hepatitis or in transplants ofheterologous tissue such as bone marrow, can be engineered usingimmune-suppressive viral gene products which suppress surface expressionof transplantation (MHC) antigen. Group C adenoviruses Ad2 and Ad5possess a 19 kd glycoprotein (gp 19) encoded in the E3 region of thevirus. This gp 19 molecule binds to class I MHC molecules in theendoplasmic reticulum of cells, and prevents terminal glycosylation andtranslocation of class I MHC to the cell surface. For example, prior tobone marrow transplantation, donor bone marrow cells may be infectedwith gp 19-encoding vector constructs which, upon expression of the gp19, inhibit the surface expression of MHC class I transplantationantigens. These donor cells may be transplanted with low risk of graftrejection and may require a minimal immunosuppressive regimen for thetransplant patient. This may allow an acceptable donor-recipientchimeric state to exist with fewer complications. Similar treatments maybe used to treat the range of so-called autoimmune diseases, includinglupus erythromiatis, multiple sclerosis, rheumatoid arthritis or chronichepatitis B infection.

An alternative method involves the use of anti-sense message, ribozyme,or other specific gene expression inhibitor specific for T cell cloneswhich are autoreactive in nature. These block the expression of the Tcell receptor of particular unwanted clones responsible for anautoimmune response. The anti-sense, ribozyme, or other gene may beintroduced using the viral vector delivery system.

7. Replacement or Augmentation Gene Therapy

One further aspect of the present invention relates to transformingcells of an animal with recombinant alphavirus vectors which serve asgene transfer vehicles to supply genetic sequences capable of expressinga therapeutic protein. Within one embodiment of the present invention,the viral vector construct is designed to express a therapeutic proteincapable of preventing, inhibiting, stabilizing or reversing an inheritedor noninherited genetic defect in metabolism, immune regulation,hormonal regulation, enzymatic or membrane associated structuralfunction. This embodiment also describes the viral vector capable oftransducing individual cells, whereby the therapeutic protein is able tobe expressed systemically or locally from a specific cell or tissue,whereby the therapeutic protein is capable of (a) the replacement of anabsent or defective cellular protein or enzyme, or (b) supplementproduction of a defective of low expressed cellular protein or enzyme.Such diseases may include cystic fibrosis, Parkinson's disease,hypercholesterolemia, adenosine deaminase deficiency, β-globindisorders, Hemophilia A & B, Gaucher's disease, diabetes and leukemia.

As an example of the present invention, a recombinant alphavirus viralvector can be used to treat Gaucher disease. Briefly, Gaucher disease isa genetic disorder that is characterized by the deficiency of the enzymeglucocerebrosidase. This type of therapy is an example of a single genereplacement therapy by providing a functional cellular enzyme. Thisenzyme deficiency leads to the accumulation of glucocerebroside in thelysosomes of all cells in the body. However, the disease phenotype ismanifested only in the macrophages, except in the very rare neuronpathicforms of the disease. The disease usually leads to enlargement of theliver and spleen and lesions in the bones. (For a review, see Science256:794, 1992, and The Metabolic Basis of Inherited Disease, 6th ed.,Scriver et al., vol. 2, p. 1677).

8. Lymphokines and Lymphokine Receptors

As noted above, the present invention provides alphavirus particleswhich can, among other functions, direct the expression of one or morecytokines or cytokine receptors.

Briefly, in addition to their role as cancer therapeutics, cytokines canhave negative effects resulting in certain pathological conditions. Forexample, most resting T-cells, B cells, large granular lymphocytes andmonocytes do not express IL-2R (receptor). In contrast to the lack ofIL-2R expression on normal resting cells, IL-2R is expressed by abnormalcells in patients with certain leukemias (ATL, Hairy-cell, Hodgkins,acute and chronic granulocytic), autoimmune diseases, and is associatedwith allograft rejection. Interestingly, in most of these patients theserum concentration of a soluble form of IL-2R is elevated. Therefore,with certain embodiments of the invention therapy may be effected byincreasing the serum concentration of the soluble form of the cytokinereceptor. For example, in the case of IL-2R, an alphavirus vector can beengineered to produce both soluble IL-2R and IL-2R, creating a highaffinity soluble receptor. In this configuration, serum IL-2 levelswould decrease, inhibiting the paracrine loop.

This same strategy may also be effective against autoimmune diseases. Inparticular, because some autoimmune diseases (e.g., Rheumatoidarthritis, SLE) are also associated with abnormal expression of IL-2,blocking the action of IL-2 by increasing the serum level of receptormay also be utilized in order to treat such autoimmune diseases.

In other cases inhibiting the levels of IL-1 may be beneficial. Briefly,IL-1 consists of two polypeptides, IL-1 and IL-1, each of which hasplieotropic effects. IL-1 is primarily synthesized by mononuclearphagocytes, in response to stimulation by microbial products orinflammation. There is a naturally occurring antagonist of the IL-1R,referred to as the IL-1 Receptor antagonist (“IL-1Ra”). This IL-1Rantagonist has the same molecular size as mature IL-1 and isstructurally related to it. However, binding of IL-1Ra to the IL-1R doesnot initiate any receptor signaling. Thus, this molecule has a differentmechanism of action than a soluble receptor, which complexes with thecytokine and thus prevents interaction with the receptor. IL-1 does notseem to play an important role in normal homeostasis. In animals,antibodies to IL-1 receptors reduce inflammation and anorexia due toendotoxins and other inflammation inducing agents.

In the case of septic shock, IL-1 induces secondary compounds which arepotent vasodilators. In animals, exogenously supplied IL-1 decreasesmean arterial pressure and induces leukopenia. Neutralizing antibody toIL-1 reduced endotoxin-induced fever in animals. In a study of patientswith septic shock who were treated with a constant infusion of IL-1R forthree days, the 28 day mortality was 16% compared to 44% in patients whoreceived placebo infusions.

In the case of autoimmune disease, reducing the activity of IL-1 reducesinflammation. Similarly, blocking the activity of IL-1 with recombinantreceptors can result in increased allograft survival in animals, againpresumably by decreasing inflammation.

These diseases provide further examples where alphavirus vectors may beengineered to produce a soluble receptor or more specifically the IL-1Ramolecule. For example, in patients undergoing septic shock, a singleinjection of IL-1Ra producing vector particles could replace the currentapproach requiring a constant infusion of recombinant IL-1R.

Cytokine responses, or more specifically, incorrect cytokine responsesmay also be involved in the failure to control or resolve infectiousdiseases. Perhaps the best studied example is non-healing forms ofleishmaniasis in mice and humans which have strong, butcounterproductive T_(H)2-dominated responses. Similarly, lepromotomatousleprosy is associated with a dominant, but inappropriate T_(H)2response. In these conditions, alphavirus-based gene therapy may beuseful for increasing circulating levels of IFN gamma, as opposed to thesite-directed approach proposed for solid tumor therapy. IFN gamma isproduced by T_(H)-1 T-cells, and functions as a negative regulator ofT_(H)-2 subtype proliferation. IFN gamma also antagonizes many of theIL-4 mediated effects on B-cells, including isotype switching to IgE.

IgE, mast cells and eosinophils are involved in mediating allergicreaction. IL-4 acts on differentiating T-cells to stimulate T_(H)-2development, while inhibiting T_(H)-1 responses. Thus, alphavirus-basedgene therapy may also be accomplished in conjunction with traditionalallergy therapeutics. One possibility is to deliver alphavirus-IL4R withsmall amounts of the offending allergen (i.e., traditional allergyshots). Soluble IL-4R would prevent the activity of IL-4, and thusprevent the induction of a strong T_(H)-2 response.

9. Suicide Vector

One further aspect of the present invention relates to the expression ofalphavirus suicide vectors to limit the spread of wild-type alphavirusin the packaging/producer cell lines. Briefly, within one embodiment thealphavirus suicide vector would be comprised of an antisense or ribozymesequence, specific for the wild-type alphavirus sequence generated froman RNA recombination event between the 3′ sequences of the junctionregion of the vector, and the 5′ alphavirus structural sequences of thepackaging cell line expression vector. The antisense or ribozymemolecule would only be thermostable in the presence of the specificrecombination sequence and would not have any other effect in thealphavirus packaging/producer cell line. Alternatively, a toxic molecule(such as those disclosed below), may also be expressed in the context ofa vector that would only express in the presence of wild-typealphavirus.

10. Alphavirus Vectors to Prevent the Spread of Metastatic Tumors

One further aspect of the present invention relates to the use ofalphavirus vectors for inhibiting or reducing the invasiveness ofmalignant neoplasms. Briefly, the extent of malignancy typically relatesto vascularization of the tumor. One cause for tumor vascularization isthe production of soluble tumor angiogenesis factors (TAF) (Paweletz etal., Crit. Rev. Oncol. Hematol. 9:197, 1989) expressed by some tumors.Within one aspect of the present invention, tumor vascularization may beslowed by using alphavirus vectors to express antisense or ribozyme RNAmolecules specific for TAF. Alternatively, anti-angiogenesis factors(Moses et al., Science 248:1408, 1990; Shapiro et al., PNAS 84:2238,1987) may be expressed either alone or in combination with theabove-described ribozymes or antisense sequences in order to slow orinhibit tumor vascularization. Alternatively, alphavirus vectors canalso be used to express an antibody specific for the TAF receptors onsurrounding tissues.

11. Administration of Alphavirus Particles

Within other aspects of the present invention, methods are provided foradministering recombinant alphavirus vectors or particles. Briefly, thefinal mode of viral vector administration usually relies on the specifictherapeutic application, the best mode of increasing vector potency, andthe most convenient route of administration. Generally, this embodimentincludes recombinant alphavirus vectors which can be designed to bedelivered by, for example, (1) direct injection into the blood stream;(2) direct injection into a specific tissue or tumor; (3) oraladministration; (4) nasal inhalation; (5) direct application to mucosaltissues; or (6) ex vivo administration of transduced autologous cellsinto the animal. Thus the therapeutic alphavirus vector can beadministered in such a fashion such that the vector can (a) transduce anormal healthy cell and transform the cell into a producer of atherapeutic protein or agent which is secreted systemically or locally,(b) transform an abnormal or defective cell, transforming the cell intoa normal functioning phenotype, (c) transform an abnormal cell so thatit is destroyed, and/or (d) transduce cells to manipulate the immuneresponse.

I. MODULATION OF TRANSCRIPTION FACTOR ACTIVITY

In yet another embodiment, alphavirus vectors may be utilized in orderto regulate the growth control activity of transcription factors in theinfected cell. Briefly, transcription factors directly influence thepattern of gene expression through sequence-specific trans-activation orrepression (Karin, New Biologist 21:126-131, 1990). Thus, it is notsurprising that mutated transcription factors represent a family ofoncogenes. Alphavirus gene transfer therapy can be used, for example, toreturn control to tumor cells whose unregulated growth is activated byoncogenic transcription factors, and proteins which promote or inhibitthe binding cooperatively in the formation of homo- and heterodimertarns-activating or repressing transcription factor complexes.

One method for reversing cell proliferation would be to inhibit thetrans-activating potential of the c-myc/Max heterodimer transcriptionfactor complex. Briefly, the nuclear oncogene c-myc is expressed byproliferating cells and can be activated by several distinct mechanisms,including retroviral insertion, amplification, and chromosomaltranslocation. The Max protein is expressed in quiescent cells and,independently of c-myc, either alone or in conjunction with anunidentified factor, functions to repress expression of the same genesactivated by the myc/Max heterodimer (Cole, Cell 65:715-716, 1991).

Inhibition of c-myc or c-myc/Max proliferation of tumor cells may beaccomplished by the overexpression of Max in target cells controlled byalphavirus vectors. The Max protein is only 160 amino acids(corresponding to 480 nucleotide RNA length) and is easily incorporatedinto an alphavirus vector either independently, or in combination withother genes and/or antisense/ribozyme moieties targeted to factors whichrelease growth control of the cell.

Modulation of homo/hetero-complex association is another approach tocontrol transcription factor activated gene expression. For example,transport from the cytoplasm to the nucleus of the trans-activatingtranscription factor NF-B is prevented while in a heterodimer complexwith the inhibitor protein IB. Upon induction by a variety of agents,including certain cytokines, IB becomes phosphorylated and NF-B isreleased and transported to the nucleus, where it can exert itssequence-specific trans-activating function (Baeuerle and Baltimore,Science 242:540-546, 1988). The dissociation of the NF-B/IB complex canbe prevented by masking with an antibody the phosphorylation site of IB.This approach would effectively inhibit the trans-activation activity ofthe NF-IB transcription factor by preventing its transport to thenucleus. Expression of the IB phosphorylation site specific antibody orprotein in target cells may be accomplished with an alphavirus genetransfer vector. An approach similar to the one described here could beused to prevent the formation of the trans-activating transcriptionheterodimer factor AP-1 (Turner and Tijan, Science 243:1689-1694, 1989),by inhibiting the association between the jun and fos proteins.

J. PHARMACEUTICAL COMPOSITIONS

As noted above, the present invention also provides pharmaceuticalcompositions comprising a recombinant Sindbis particle or virus, orSindbis vector construct, in combination with a pharmaceuticallyacceptable carrier, diluent, or recipient.

Briefly, infectious recombinant virus (also referred to above asparticles) may be preserved either in crude or purified forms. In orderto produce virus in a crude form, virus-producing cells may first becultivated in a bioreactor, wherein viral particles are released fromthe cells into the culture media. Virus may then be preserved in crudeform by first adding a sufficient amount of a formulation buffer to theculture media containing the recombinant virus to form an aqueoussuspension. Within certain preferred embodiments, the formulation bufferis an aqueous solution that contains a saccharide, a high molecularweight structural additive, and a buffering component in water. Theaqueous solution may also contain one or more amino acids.

The recombinant virus can also be preserved in a purified form. Morespecifically, prior to the addition of the formulation buffer, the cruderecombinant virus described above may be clarified by passing it througha filter and then concentrated, such as by a cross flow concentratingsystem (Filtron Technology Corp., Nortborough, Mass). Within oneembodiment, DNase is added to the concentrate to digest exogenous DNA.The digest is then diafiltrated in order to remove excess mediacomponents and to establish the recombinant virus in a more desirablebuffered solution. The diafiltrate is then passed over a Sephadex S-500gel column and a purified recombinant virus is eluted. A sufficientamount of formulation buffer is then added to this eluate in order toreach a desired final concentration of the constituents and to minimallydilute the recombinant virus. The aqueous suspension may then be stored,preferably at −70° C., or immediately dried. As above, the formulationbuffer may be an aqueous solution that contains a saccharide, a highmolecular weight structural additive, and a buffering component inwater. The aqueous solution may also contain one or more amino acids.

Crude recombinant virus may also be purified by ion exchange columnchromatography. Briefly, crude recombinant virus may be clarified byfirst passing it through a filter, followed by loading the filtrate ontoa column containing a highly sulfonated cellulose matrix. Therecombinant virus may then be eluted from the column in purified form byusing a high salt buffer, and the high salt buffer exchanged for a moredesirable buffer by passing the eluate over a molecular exclusioncolumn. A sufficient amount of formulation buffer is then added, asdiscussed above, to the purified recombinant virus and the aqueoussuspension is either dried immediately or stored, preferably at −70° C.

The aqueous suspension in crude or purified form can be dried bylyophilization or evaporation at ambient temperature. Briefly,lyophilization involves the steps of cooling the aqueous suspensionbelow the gas transition temperature or below the eutectic pointtemperature of the aqueous suspension, and removing water from thecooled suspension by sublimation to form a lyophilized virus. Within oneembodiment, aliquots of the formulated recombinant virus are placed intoan Edwards Refrigerated Chamber (3 shelf RC3S unit) attached to a freezedryer (Supermodulyo 12K). A multistep freeze drying procedure asdescribed by Phillips et al. (Cryobiology 18:414, 1981) is used tolyophilize the formulated recombinant virus, preferably from atemperature of −40° C. to −45° C. The resulting composition containsless than 10% water by weight of the lyphilized virus. Once lyophilized,the recombinant virus is stable and may be stored at −20° C. to 25° C.,as discussed in more detail below.

Within the evaporative method, water is removed from the aqueoussuspension at ambient temperature by evaporation. Within one embodiment,water is removed through spray-drying (EP 520,748). Within thespray-drying process, the aqueous suspension is delivered into a flow ofpreheated gas, usually air, whereupon water rapidly evaporates fromdroplets of the suspension. Spray-drying apparatus are available from anumber of manufacturers (e.g., Drytec, Ltd., Tonbridge, England;Lab-Plant, Ltd. Huddersfield, England). Once dehydrated, the recombinantvirus is stable and may be stored at −20° C. to 25° C. Within themethods described herein, the resulting moisture content of the dried orlyophilized virus may be determined through use of a Karl-Fischerapparatus (EM Science Aquastar' V1B volumetric titrator, Cherry Hill,N.J.), or through a gravimetric method.

The aqueous solutions used for formulation, as previously described, arepreferably composed of a saccharide, high molecular weight structuraladditive, a buffering component, and water. The solution may alsoinclude one or more amino acids. The combination of these components actto preserve the activity of the recombinant virus upon freezing andlyophilization or drying through evaporation. Although a preferredsaccharide is lactose, other saccharides may be used, such as sucrose,mannitol, glucose, trehalose, inositol, fructose, maltose or galactose.In addition, combinations of saccharides can be used, for example,lactose and mannitol, or sucrose and mannitol. A particularly preferredconcentration of lactose is 3%-4% by weight. Preferably, theconcentration of the saccharide ranges from 1% to 12% by weight.

The high molecular weight structural additive aids in preventing viralaggregation during freezing and provides structural support in thelyophilized or dried state. Within the context of the present invention,structural additives are considered to be of “high molecular weight” ifthey are greater than 5000 m.w. A preferred high molecular weightstructural additive is human serum albumin. However, other substancesmay also be used, such as hydroxyethyl-cellulose,hydroxymethyl-cellulose, dextran, cellulose, gelatin, or povidone. Aparticularly preferred concentration of human serum albumin is 0.1% byweight. Preferably, the concentration of the high molecular weightstructural additive ranges from 0.1% to 10% by weight.

The amino acids, if present, function to further preserve viralinfectivity upon cooling and thawing of the aqueous suspension. Inaddition, amino acids function to further preserve viral infectivityduring sublimation of the cooled aqueous suspension and while in thelyophilized state. A preferred amino acid is arginine, but other aminoacids such as lysine, ornithine, serine, glycine, glutamine, asparagine,glutamic acid or aspartic acid can also be used. A particularlypreferred arginine concentration is 0.1% by weight. Preferably, theamino acid concentration ranges from 0.1% to 10% by weight.

The buffering component acts to buffer the solution by maintaining arelatively constant pH. A variety of buffers may be used, depending onthe pH range desired, preferably between 7.0 and 7.8. Suitable buffersinclude phosphate buffer and citrate buffer. A particularly preferred pHof the recombinant virus formulation is 7.4, and a preferred buffer istromethamine.

In addition, it is preferable that the aqueous solution contain aneutral salt which is used to adjust the final formulated recombinantalphavirus to an appropriate iso-osmotic salt concentration. Suitableneutral salts include sodium chloride, potassium chloride or magnesiumchloride. A preferred salt is sodium chloride.

Aqueous solutions containing the desired concentration of the componentsdescribed above may be prepared as concentrated stock solutions.

It will be evident to those skilled in the art, given the disclosureprovided herein, that it may be preferable to utilize certainsaccharides within the aqueous solution when the lyophilized virus isintended for storage at room temperature. More specifically, it ispreferable to utilize disaccharides, such as lactose or trehalose,particularly for storage at room temperature.

The lyophilized or dehydrated viruses of the subject invention may bereconstituted using a variety of substances, but are preferablyreconstituted using water. In certain instances, dilute salt solutionswhich bring the final formulation to isotonicity may also be used. Inaddition, it may be advantageous to use aqueous solutions containingcomponents known to enhance the activity of the reconstituted virus.Such components include cytokines, such as IL-2, polycations, such asprotamine sulfate, or other components which enhance the transductionefficiency of the reconstituted virus. Lyophilized or dehydratedrecombinant virus may be reconstituted with any convenient volume ofwater or the reconstituting agents noted above that allow substantial,and preferably total solubilization of the lyophilized or dehydratedsample.

The following examples are offered by way of illustration, and not byway of limitation.

EXAMPLES Example 1 Cloning of a Sindbis Genomic Length cDNA

The nature of viruses having an RNA genome of positive polarity is suchthat, when introduced into a eukaryotic cell which serves as apermissive host, the purified genomic nucleic acid serves as afunctional message RNA (mRNA) molecule for translation of the viralreplicase proteins. Therefore, this genomic RNA, purified from thevirus, can initiate the same infection cycle that is characteristic ofinfection by the wild-type virus from which the RNA was purified.

For example, Sindbis virus strain AR339 (ATCC #VR-1248, Taylor et al.,Am. J. Trop. Med. Hyg. 4:844 1955; isolated from the mosquito Culexusunivittatus) is propagated in baby hamster kidney (BHK-21) cells (ATCC#CCL-10), infected at low multiplicity (0.1 PFU/cell). Alternatively,another HR-derived Sindbis virus strain, obtained from Lee Biomolecular(Sand Diego, Calif.), also is used and propagated by the same methods.Sindbis virions are precipitated from a clarified lysate at 48 hourspost-infection, with 10% (w/v) of polyethylene glycol (PEG-8000) at 0°C., as described previously. Sindbis virions which are contained in thePEG pellet are then lysed with 2% SDS, and the polyadenylated mRNAisolated by chromatography utilizing commercially available oligo-dTcolumns (Invitrogen, San Diego, Calif.).

Two rounds of first strand cDNA synthesis are performed on the polyAselected mRNA, using an oligonucleotide primer with the sequence shownbelow:

5′-TATATTCTAGA(dT)₂₅-GAAATG-3′  (SEQ. ID NO. 3)

Briefly, this primer contains at its 5′ end, a five nucleotide ‘buffersequence’ for efficient restriction endonuclease digestion, followed bythe Xba I recognition sequence, 25 consecutive dT nucleotides and sixnucleotides which are precisely complementary to the extreme Sindbis 3′end. Thus, selection for first round cDNA synthesis occurs at twolevels: (1) polyadenylated molecules, a prerequisite for functionalmRNA, and (2) selective priming from Sindbis mRNA molecules, in a poolpossibly containing multiple mRNA species. Further, the reversetranscription is performed in the presence of 10 mM MeHgOH to mitigatethe frequency of artificial stops during reverse transcription.

Primary genomic length Sindbis cDNA is then amplified by PCR in sixdistinct segments using six pairs of overlapping primers. Briefly, inaddition to viral complementary sequences, the Sindbis 5′ end forwardprimer is constructed to contain a 19 nucleotide sequence correspondingto the bacterial SP6 RNA polymerase promoter and the Apa I restrictionendonuclease recognition sequence linked to its 5′ end. The bacterialSP6 RNA polymerase is poised such that transcription in vitro results inthe inclusion of only a single non-viral G ribonucleotide linked to theA ribonucleotide, which corresponds to the authentic Sindbis 5′ end.Inclusion of the Apa I recognition sequence facilitates insertion of thePCR amplicon into the plasmid vector (pKS II+, Stratagene, San Diego,Calif.) polylinker sequence. A five nucleotide ‘buffer sequence’ is alsoinserted prior to the Apa I recognition sequence in order to permitefficient digestion. The sequence of the SP6-5′ Sindbis forward primerand all of the primer pairs necessary to amplify the entire Sindbisgenome are shown below. (Note that “nt” and “nts” as utilizedhereinafter refer to “nucleotide” and “nucleotides,” respectively). Thereference sequence (GenBank accession no. SINCG) is from Strauss et al.,Virology 133:92-110.

Seq. Recognition Primer Location ID No. Sequence Sequence (5′->3′)SP6-1A Apa I/SP6/+ SIN nts.1-18 4 TATATGGGCCCGATTTAGGTGAC Apa IACTATAGATTGACGGCGTAGTAC AC 1B 3182-3160 5 CTGGCAACCGGTAAGTACGATAC Age I2A 3144-3164 6 ATACTAGCCACGGCCGGTATC Age I 2B 5905-5885 7TCCTCTTTCGACGTGTCGAGC Eco RI 3A 5844-5864 8 ACCTTGGAGCGCAATGTCCTG Eco RI7349R 7349-7328 9 CCTTTTCAGGGGATCCGCCAC Bam HI 7328F 7328-7349 10GTGGCGGATCCCCTGAAAAGG Bam HI 3B 9385-9366 11 TGGGCCGTGTGGTCGTCATG Bcl I4A 9336-9356 12 TGGGTCTTCAACTCACCGGAC Bcl I 10394R 10394-10372 13CAATTCGACGTACGCCTCACTC Bsi WI 10373F 10373-10394 14GAGTGAGGCGTACGTCGAATTG Bsi WI 4B Xba I/dT₂₅/ 11703-11698 3TATATTCTAGA(dT)₂₅-GAAATG Xba I

PCR amplification of Sindbis cDNA with the six primer sets shown aboveis performed in separate reactions, using the THERMALASE™ thermostableDNA polymerase (Amresco Inc., Solon, Ohio) and the buffer containing 1.5mM MgCl₂, provided by the supplier. Additionally, the reactions contain5% DMSO, and the HOT START WAX™ beads (Perkin-Elmer), using the PCRamplification protocol shown below:

Temperature (° C.) Time (Min.) No. Cycles 94 2 1 94 0.5 55 0.5 35 72 3.572 10 10

Following amplification, the six reaction products are inserted firstinto the pCR II vector (Invitrogen), then using the appropriate enzymesshown above, are inserted, stepwise, into the pKS II+ (Stratagene)vector, between the Apa I and Xba I sites. This clone is designated aspVGSP6GEN.

The Sindbis genomic cDNA clone pVGSP6GEN is linearized by digestion withXba I, which cuts pVGSP6GEN once, immediately adjacent and downstream ofthe 25 nucleotide long poly dA:dT stretch. The linearized pVGSP6GENclone is purified with GENECLEAN™ (BIO 101, La Jolla, Calif.), andadjusted to a concentration of 0.5 mg/ml. Transcription of thelinearized pVGSP6GEN clone is performed in vitro at 40° C. for 90minutes according to the following reaction conditions: 2 ul DNA/4.25 ulH₂O; 10 ul 2.5 mM NTPs (UTP, ATP, GTP, CTP); 1.25 ul 20 mMMe⁷G(5′)ppp(5′)G cap analogue; 1.25 ul 100 mM DTT; 5 ul 5X transcriptionbuffer (Promega, Madison, Wis.); 0.5 ul RNasin (Promega); 0.25 ul 10mg/ml bovine serum albumin; and 0.5 ul SP6 RNA polymerase (Promega). Thein vitro transcription reaction products can be digested with DNase I(Promega) and are purified by sequential phenol/CHCl₃ and etherextraction, followed by ethanol precipitation, or alternatively, can beused directly for transfection. The in vitro transcription reactionproducts or purified RNA are complexed with a commercial cationic lipidcompound (for example, LIPOFECTIN™, GIBCO-BRL, Gaithersburg, Md.), andapplied to BHK-21 cells maintained in a 60 mM petri dish at 75%confluency. The transfected cells are incubated at 30° C. After 94 hourspost-transfection, extensive cytopathologic effects (CPE) are observed.No obvious CPE is observed in plates not receiving RNA transcribed fromthe Sindbis cDNA clone. Further, 1 ml of supernatant taken fromtransfected cells, added to fresh monolayers of BHK-21 cells, andincubated at 30° C. or 37° C. results in obvious CPE within 18 hours.This demonstrates that the Sindbis cDNA clone pVGSP6GEN is indeedinfectious.

Sequence analysis of pVGSP6GEN, shown in Table 1, reveals multiplesequence differences between the Sindbis genomic clone described herein,and the viral clone sequence provided in Genbank (GenBank Accession No.SINCG). Many sequence differences result in the substitution ofnon-conservative amino acids changes in the Sindbis proteins. To addresswhich sequence changes are unique to the virus strain used for cloning,as described herein, or are a result of cloning artifact, virion RNA isamplified by RT-PCR as described above, and sequence relating to thenucleotides in question is determined by direct sequencing of the RT-PCRamplicon product, using a commercially available kit (Promega, Madison,Wis.), and compared to the corresponding pVGSP6GEN sequence. The resultsof this study are given in Table 2. Briefly, three non-conservativeamino acid changes, Gly→Glu, Asp→Gly, and Tyr→Cys, which are a result ofcloning artifact are observed respectively at viral nucleotides 2245,6193, and 6730. These nucleotide changes resulting in non-conservativeamino changes all map to the viral non-structural protein (NSP) genes,nt 2245 to NSP 2, and nts 6193 and 6730 to NSP4.

Repair of the NSP 2 and NSP 4 genes is accomplished by RT-PCR, asdescribed above, using virion RNA from a 5 times plaque purified stock.The SP6-1A/1B primer pair described above is used to repair the nt 2245change. The RT-PCR amplicon product is digested with Eco 47III and BglII, and the 882 bp fragment is purified by 1% agarose/TBE gelelectrophoresis, and exchanged into the corresponding region of thepVGSP6GEN clone, prepared by digestion with Eco 47III and Bgl II, andtreatment with CIAP. The 3A/7349R primer pair described above is used torepair the nt 6193 and nt 6730 changes. The RT-PCR amplicon product isdigested with Eco RI and Hpa I, and the 1,050 bp fragment is purified by1% agarose/TBE gel electrophoresis, and exchanged into the correspondingregion of the pVGSP6GEN clone. This clone is designated pVGSP6GENrep.Transfection of BHK cells with in vitro transcribed RNA frompVGSP6GENrep DNA, linearized by digestion with Xba I as described above,results in extensive CPE within 18 hours post-transfection.

TABLE 1 SINDBIS GENOMIC CLONE DIFFERENCES BETWEEN pVGSP6GEN AND GENBANKSEQUENCES Location amino acid SIN nt # Change Codon Change in Codonchange Noncoding Region: 45 T → C N.A. N.A. N.A. Non-structuralProteins: 353 C → T UAU → UAC 3′ Tyr → Tyr 1095 A → C AUA → CUA 1′ Ile →Leu 1412 T → C UUU → UUC 3′ Phe → Phe 2032 A → G GAG → GGG 2′ Glu → Gly2245 G → A GGG → GAG 2′ Gly → Glu 2258 A → C UCA → UCC 3′ Ser → Ser 2873A → G CAA → CAG 3′ Gln → Gln 2992 C → T CCC → CUC 2′ Pro → Leu 3544 T →C GUC → GCC 2  Val → Leu 3579 A → G AAA → GAA 1′ Leu → Glu 3822 A → GACC → GCC 1′ Thr → Ala 3851 T → C CUU → CUC 3′ Leu → Leu 5351 A → T CAA→ CAU 3′ Gln → His 5466 G → A GGU → AGU 1′ Gly → Ser 5495 T → C AUU →AUC 3′ Ile → Ile 5543 A → T ACA → ACU 3′ Thr → Thr 5614 T → C GUA → GCA2′ Val → Ala 6193 A → G GAC → GGC 2′ Asp → Gly 6564 G → A GCA → ACA 1′Ala → Thr 6730 A → G UAC → UGC 2′ Tyr → Cys Structural Proteins: 8637 A→ G AUU → GUU 1′ Ile → Val 8698 T → A GUA → GAA 2′ Val → Glu 9108 AAGdel AAG → del 1′-3′ Glu → del 9144 A → G AGA → GGA 1′ Arg → Gly 9420 A →G AGU → GGU 1′ Ser → Gly 9983 T → G GCU → GCG 3′ Ala → Ala 10469 T → AAUU → AUA 3′ Ile → Ile 10664 T → C UUU → UUC 3′ Phe → Phe 10773 T → GUCA → GCA 1′ Ser → Ala

TABLE 2 SINDBIS GENOMIC CLONE ARTIFACT ANALYSIS Amino Acid pVGSP6GENCloning SIN nt # change Unique Artifact Nonstructural Proteins: 2032 Glu→ Gly +* 2245 Gly → Glu + 2258 Ser → Ser +* 2873 Gln → Gln + 2992 Pro →Leu + 3544 Val → Leu + 3579 Leu → Glu + 3822 Thr → Ala + 3851 Leu →Leu + 5351 Gln → His + 5466 Gly → Ser + 5495 Ile → Ile + 5543 Thr →Thr + 6193 Asp → Gly + 6730 Tyr → Cys + Structural Proteins: 8637 Ile →Val + 8698 Val → Glu + 9108 Glu → del + 9144 Arg → Gly + *Mixture: BothGenbank and pVGSP6GEN Sindbis Strains present at this nucleotide.

Example 2 Generation of DNA Vectors Which Initiate Alphavirus Infection;Eukaryotic Layered Vector Initiation Systems

As noted above, the present invention provides eukaryotic layered vectorinitiation systems which generally comprise a promoter which is capableof initiating the 5′ synthesis of RNA from cDNA, a construct which iscapable of autonomous or autocatalytic replication in a cell, theconstruct also being capable of expressing a heterologous nucleic acidsequence, and a 3′ sequence which controls transcription termination.Within one embodiment, such constructs may be constructed of thefollowing ordered elements: a 5′ eukaryotic promoter capable ofinitiating the synthesis of viral RNA at the authentic alphavirus 5′end, a 5′ sequence which is capable of initiating transcription of analphavirus, a nucleotide sequence encoding alphavirus non-structuralproteins, a viral junction region, a heterologous sequence, analphavirus RNA polymerase recognition sequence, and a 3′ transcriptiontermination/polyadenylation signal sequence. Such alphavirus cDNAexpression vectors may also include intervening sequences (introns),which are spliced from the pre-RNA in the nucleus prior to transport tothe cytoplasm, and which may improve the overall efficiency of thesystem, in terms of molecules of functional mRNA transported to thecytoplasm/nuclear DNA template. The intron splicing signals are located,for example, between Sindbis and heterologous gene regions as describedin Example 3.

Construction of a eukaryotic layered vector initiation system utilizingthe Sindibis clone pVGSP6GENrep and mammalian RNA polymerase IIpromoters is accomplished as follows. Briefly, plasmid pVGSP6GEN rep isdigested with BglII and Xba I, and the reaction products areelectrophoresed on a 0.8% agarose/TBE gel. The resulting 9,438 bpfragment is excised, purified with GENECLEAN®, and ligated into the4,475 bp vector fragment resulting from treatment of pCDNA3 (Invitrogen)with Bgl II, Xba I, and CIAP. This construction is designated aspcDNASINbgl/xba.

The U3 region of the long terminal repeat (LTR) from Moloney murineleukemia virus (Mo-MLV) is positioned at the 5′ viral end such that thefirst transcribed nucleotide is a single G residue, which is capped invivo, followed by the Sindbis 5′ end. Juxtaposition of the Mo-MLV LTRand the Sindbis 5′ end is accomplished by overlapping PCR as describedbelow. Amplification of the Mo-MLV LTR in the first primary PCR reactionis accomplished in a reaction containing the BAG vector (Price et al.,PNAS 84: 156-160, 1987) and the following primer pair.

Forward primer: BAGBgl2F1 (buffer sequence/BglII recognitionsequence/Mo-MLV LTR nts 1-22):

5′-TATATAGATCTAATGAAAGACCCCACCTGTAGG   (SEQ. ID NO. 15)

Reverse primer: BAGwt441R2 (SIN nts 5-1/Mo-MLV LTR nts 441-406):

5′-TCAATCCCCGAGTGAGGGGTTGTGGGCTCTTTTATTGAGC   (SEQ. ID NO. 16)

PCR amplification of the Mo-MLV LTR with the primer pair shown above isperformed using the THERMALASE® thermostable DNA polymerase and thebuffer containing 1.5 mM MgCl₂, provided by the supplier. Additionally,the reaction contains 5% DMSO, and the HOT START WAX® beads, using thePCR amplification protocol shown below:

Temperature (° C.) Time (Min.) No. Cycles 94 2 1 94 0.5 55 0.5 35 72 0.572 10 1

Amplification of the Sindbis 5′ end in the second primary PCR reactionis accomplished in a reaction containing the pVGSP6GENrep clone and thefollowing primer pair:

Forward primer: (Mo-MLV LTR nts 421-441/SIN nts 1-16):

5′-CCACAACCCCTCACTCGGGGATTGACGGCGTAGTAC   (SEQ. ID NO. 17)

Reverse primer: (SIN nts 3182-3160):

5′-CTGGCAACCGGTAAGTACGATAC   (SEQ. ID NO. 18)

PCR amplification of the Mo-MLV LTR is accomplished with the primer pairand amplification reaction conditions described above, utilizing the PCRamplification protocol shown below:

Temperature (° C.) Time (Min.) No. Cycles 94 2 1 94 0.5 55 0.5 35 72 3.072 10 1

The 457 bp and 3202 bp products from the primary PCR reactions arepurified with GENECLEAN®, and combined in a secondary PCR reaction withthe following primer pair:

Forward primer: BAGBgl2F1 (buffer sequence/BglII recognitionsequence/Mo-MLV LTR nts 1-22):

5′-TATATAGATCTAATGAAAGACCCCACCTGTAGG   (SEQ. ID NO. 15)

Reverse primer: (SIN nts 2300-2278):

5′-GGTAACAAGATCTCGTGCCGTG   (SEQ. ID NO.19)

PCR amplification of the primer PCR amplicon products is accomplishedutilizing the primer pair and amplification reaction conditions shownabove, and using the following PCR amplification protocol:

Temperature (° C.) Time (Min.) No. Cycles 94 2 1 94 0.5 55 0.5 35 72 3.072 10 1

The 25 3′ terminal bases of the first primary PCR amplicon productoverlaps with the 25 5′ terminal bases of the second primary PCRamplicon product; the resultant 2,752 bp overlapping secondary PCRamplicon product is purified by 0.8% agarose/TBE electrophoresis,digested with Blg II, and the 2,734 bp product is ligated intopcDNASINbgl/xba treated with Bgl II and CIAP. The resulting constructionis 16,656 bps and is designated pVGELVIS. The sequence of pVGELVIS isgiven in FIG. 3 (SEQ. ID NO. 1). Sindbis nucleotides are containedwithin bases 1-11,700 of the sequence.

pVGELVIS plasmid DNA is complexed with LIPOFECTAMINE® (GIBCO-BRL,Gaithersburg, Md.) according to the conditions suggested by the supplier(ca. 5 ug DNA/8 ug lipid reagent) and added to 35 mm wells containingBHK-21 cells at approximately 75% confluency. Cytopathic effects (CPE),characteristic of wild type Sindbis virus infection are observed within48 hours post-infection. Addition of 1 ml of transfection supernatant tofresh BHK-21 monolayers results in CPE within 16 hrs. This datademonstrates the correct juxtaposition of viral cDNA and RNA polymeraseII expression cassette signals in the pVGELVIS construct, resulting inthe de novo initiation of an RNA virus from a DNA expression module.

In order to determine the relative efficiency of the pVGELVIS plasmidDNA to initiate infection characteristic of wild type Sindbis virusafter transfection into BHK cells, an infectious centers assay isperformed. Briefly, 5 ug of pVGELVIS plasmid DNA is transfected intoBHK-21 cells in 35 mm wells as described above, and at 1.5 hours posttransfection the cells are trypsinized and serially diluted 10,000-fold,over 10-fold increments, into 5×10⁵ untreated BHK cells. Thistransfected and untreated BHK cell mixture is then added to 35 mm wells.The cells are allowed to attach to the plate, and subsequently overlayedwith media containing 1.0% Noble Agar. At 48 hrs post transfection,plaques due to cell lysis (as a result of Sindbis virus replication) arevisualized either directly or after overlaying with a second layercontaining Neutral Red Stain. This experiment reveals that theefficiency of the pVGELVIS plasmid in generating wild type Sindbis virusafter transfection onto BHK cells is approximately 1×10⁴PFU/ug ofplasmid DNA.

Example 3 Preparation of RNA and DNA Alphavirus Vectors

A. CONSTRUCTION OF THE SINDBIS BASIC VECTOR

A first step in the construction of the Sindbis Basic Vector is thegeneration of two plasmid subclones containing separate elements fromthe viral 5′ and 3′ ends. These elements may then be utilized in orderto subsequently assemble a basic gene transfer vector.

Briefly, the first plasmid subclone is constructed to contain the 40terminal nucleotides of the viral 3′ end and a 25 base pair stretch ofconsecutive dA:dT nucleotides. In particular, the followingoligonucleotide pairs are first synthesized:

Forward Primer: SIN11664F: (buffer sequence/Not I site/SIN nts11664-11698):

5′-TATATATATATGCGGCCGCTTTCTTTTATTAATCAACAAAATTTTGTTTTTAA   (SEQ. IDNO.20)

Reverse Primer: SINSac11700R (buffer sequence/Sac I site dT25/SIN nts11700-11692):

5′-TATATGAGCTCTTTTTTTTTTTTTTTTTTTTTTTTTGAAATGTTAAAA   (SEQ. ID NO. 21)

The above oligonucleotides are then mixed together at equal molarconcentrations in the presence of 10 mM MgCl₂, heated to 100° C. for 5minutes and cooled slowly to room temperature. The partiallydouble-stranded molecule is then filled in using Klenow DNA polymeraseand 50 uM dNTPs. The resultant 89 bp molecule is then digested with NotI and Sac I, purified on a 2% NuSieve/1% agarose gel, and ligated intopKS II+ plasmid (Stratagene, La Jolla, Calif.), prepared by digestionwith Not I and Sac I and treatment with CIAP, at a 10:1 molar excess ofinsert:vector ratio. This construction is designated pKSII3′SIN.

The second plasmid subclone is constructed to contain the first 5′ 7,643nucleotides of Sindbis, and a bacteriophage RNA polymerase promoter ispositioned at the viral 5═ end such that only a single non-viralnucleotide is added to the authentic viral 5′ end after in vitrotranscription. Briefly, the 3′ end of this clone is derived by astandard three temperature PCR amplification with a reverse primerhaving the sequence shown below.

Reverse Primer: SINXho7643R (buffer sequence/Xho I site/SIN nts7643-7621):

5′TATATCTCGAGGGTGGTGTTGTAGTATTAGTCAG   (SEQ. ID NO. 22)

The reverse primer maps to viral nucleotides 7643-7621 and is 41 bpdownstream from the junction core element 3′ end. Additionally, viralnucleotide 7643 is 4 nucleotides upstream from the structural proteingene translation initiation codon. The first five 5′ nucleotides in thisprimer are included to serve as a ‘buffer sequence’ for the efficientdigestion of the PCR amplicon products, and are followed by 6nucleotides comprising the Xho I recognition sequence.

The forward primer in this reaction is primer 2A (described in Example1), having the following sequence:

ATACTAGCCACGGCCTGGTATC   (SEQ. ID NO. 6)

The 4510 bp amplicon product, resulting from the PCR amplification shownabove with pVGSP6GENrep plasmid (described in Example 1), as template,is digested with the enzymes Sfi I and Xho I. The resultant 2526 bpfragment is gel purified. Sindbis cDNA clone pVGSP6GENrep is alsodigested with Apa I and Sfi I, and the resultant 5144 bp fragment whichincludes the SP6 RNA polymerase promoter at its 5′ end is gel purified.The 5144 bp fragment is ligated together with the 2526 bp fragment fromabove, along with Apa I and the Xho I digested CIAP treated pKSII+plasmid. A clone is isolated having the Sindbis nucleotides 1-7643including the RNA polymerase promoter at its 5′ end contained in thepKSII+plasmid vector. This construction is designated pKSII5′SIN.

Assembly of the complete basic vector is accomplished by digestingpKSII5′SIN with Xho I and Sac I, treating with CIAP, and gel purifyingof a large 10,533 bp fragment. The 10,533 bp fragment is then ligatedtogether with a 168 bp small fragment resulting from digestion ofpKSII3′SIN with Xho I and Sac I. This resultant construction isdesignated pKSSINBV (also known as SINDBIS basic vector, see FIG. 4.)

B. CONSTRUCTION OF SINDBIS LUCIFERASE VECTOR

The firefly luciferase reporter gene is inserted into the Sindbis BasicVector in order to demonstrate the expression of a heterologous gene incells transfected with RNA that is transcribed in vitro from the Sindbisvector clone, and to demonstrate the overall functionality of theSindbis basic vector.

Construction of the Sindbis luciferase vector is performed by assemblingtogether components of 3 independent plasmids: pKSII5′SIN, pKSII3′SIN,and pGL2-basic vector. The pGL2-basic vector plasmid (Promega, Madison,Wis.) contains the entire firefly luciferase gene. Briefly, theluciferase gene is first inserted into the pKSII3′SIN plasmid. This isaccomplished by digesting pGL2 with Bam HI and Hind III, and gelpurifying a 2689 bp containing fragment. This fragment is ligated with agel purified 3008 bp large fragment resulting from digestion ofpKSII3′SIN with Bam HI and Hind III and treatment with CIAP. Theresultant construction is designated pKSII3′SIN-luc.

Final assembly of a Sindbis luciferase vector is accomplished bydigesting pKSII5′SIN with Xho I and Sac I, treating with CIAP, and gelpurifying the large 10,533 bp fragment. The pKS5′SIN 10,533 bp fragmentis ligated together with the 2854 bp small fragment resulting fromdigestion of pKSII3′SIN-luc with Xho I and Sac I. This constructioncontains the entire Sindbis nonstructural gene coding region and 3′viral elements necessary for genome replication, as well as the fireflylucierase gene positioned between these two viral 5′ and 3′ elements.This vector is designated pKSSINBV-luc (also known asSINDBIS-luciferase) and is shown schematically in FIG. 4.

C. EXPRESSION OF LUCIFERASE IN TRANSFECTED AND INFECTED BHK-21 CELLS

In order to test the functionality of the Sindbis Basic Vector, theexpression of luciferase in cells transfected with RNA transcribed invitro from Sac I-linearized pKSSINBV-luc, as described in Example 1, istested.

In addition, a complementary packaging vector, which is delected of mostof the non structural gene region, is constructed by digestion ofpVGSP6GENrep with Bsp EI and re-ligation under dilute conditions. Thisconstruction, designated pVGSP6GENdlBsp (also known as “dl Bsp EI”) isdeleted of nonstructural gene sequences between bases 422-7,054, and isshown schematically in FIG. 5. Transcription in vitro of XbaI-linearized pVGSP6GENdlBsp is as described in Example 1. Transfectionsand co-transfections are performed by complexing in vitro transcriptionproducts with LIPOFECTIN® and applying BHK-21 cells. The expression ofluciferase in transfected cells is tested 18 hours after transfection.Additionally, 1 ml of the transfection supernatant is used to infect aconfluent monolayer of BHK-21 cells and the expression of luciferase istested at 24 hours post-infection.

The results of this experiment shown in FIG. 6, demonstrate clearlyabundant reporter gene expression follows transfection of BHK-21 cellswith in vitro transcribed RNA from pKSSINBV-luc, and tranfer (e.g.,packaging) of the expression activity when cells are co-transfected within vitro transcribed RNA from pVGSP6GENdlBsp.

D. CONSTRUCTION OF ALTERED JUNCTION REGION SINDBIS VECTORS

In order to inactivate the Sindbis viral junction region, nucleotideswithin the NSP4 carboxy terminus and junction region overlap arechanged, and the vector nucleotides corresponding to Sindbis areterminated prior to the subgenomic initiation point at Sindbis nt 7598.This construction is shown schematically in FIG. 7.

Briefly, a fragment is PCR amplified from the pKSSINBV clone undernonstringent reaction cycle conditions utilizing a reverse primer havingthe following sequence:

TATATGGGCCCTTAAGACCATCGGAGCGATGCTTTATTTCCCC   (SEQ. ID NO. 23)

The underlined bases in the reverse primer relate to nucleotide changeswhich can be made in the junction region without affecting the codedamino acid (see below). All of the nucleotide changes are tranversions.

3′ end of NSP 4 (viral nts 7580-7597): TCT CTA CGG TGG TCC TAA (SEQ. IDNO. 24) ser leu arg trp ser stop (SEQ.ID NO.25)   G   C   A       T(resulting nt changes from reverse primer)

The reverse primer is complementary to Sindbis nts 7597-7566 (except atnucleotides, as shown, where junction region changes were made), andincludes at its 5′ end the 6 nucleotide Apa I recognition sequencefollowing a 5′ terminate TATAT tail ‘buffer sequence’ for efficientenzyme digestion. The forward primer in this reaction is primer 2A(described in Example 1), having the following sequence:

5′-ATACTAGCCACGGCCGGTATC   (SEQ. ID NO. 6)

The 4,464 bp amplicon resulting from a PCR reaction with pKSSINBVtemplate and using the primer pair described above is digested with SfiI and Apa I and the gel purified 2,480 bp fragment is ligated togetherwith the gel purified 5,142 bp fragment resulting from the digestion ofpKSSINBV with Apa I and Sfi I, and with the gel purified 2,961 bpfragment resulting from the digestion pKSII+ with Apa I and from thetreatment with CIAP. This construction, comprised of Sindbis nucleotides1-7597, including the changes in the junction region described above,and including the bacterial SP6 promoter attached to Sindbis nt 1 isreferred to as pKS5′SINdlJR.

Final construction of the inactivated junction region vector isaccomplished by ligation of the 7,622 bp large Sindbis fragmentresulting from digestion of pKS5═SINdlJR with Apa I, with the 3,038 bpfragment resulting from digestion of pKSII3′SIN with Apa I and treatmentwith CIAP. The positive orientation of the 5′ Sindbis element, relativeto the 3′ Sindbis element, is confirmed by restriction endonucleaseanalysis. This construction is referred to as pKSSINBVdlJR.

Initiation and synthesis of subgenomic mRNA cannot occur from thepKSSINBVdlJR vector. In order to prove this supposition, comparativeRNase protection assays using the pKSSINBV and pKSSINBVdlJR vectors areperformed. Briefly, a ³²P-end labeled RNA probe complementary in part tothe junction region, including the subgenomic RNA initiation point atviral nt 7,598 is used to hybridize with the viral RNA resulting fromthe transfection of BHK-21 cells with the pKSSINBV and pKSSINBVdlJRvectors. The RNase protection assay demonstrates that cells transfectedwith pKSSINBV have two fragments, of genomic and subgenocmi specificity,while cells transfected with pKSSINBVdlJR have only a single fragment ofgenomic specificity. These results prove that the junction region in thepKSSINBVdlJR vector is indeed inactivated.

In order to test translation of genomic RNA from the regioncorresponding to the subgenomic RNA message, the luciferase reportergene is inserted into the inactivated junction region vectorpKSSINBVdlJR described above. This construction is accomplished bydigesting the pKSSINBVdlJR with Xho I and Sac I, treating with CIAP, andgel purifying the resulting 10,197 bp fragment. The pKSSINBVdlJRfragment is ligated together with the 2854 bp small fragment resultingfrom digestion of pKSII3′SIN-luc with Xho I and Sac I. This constructioncontains the entire Sindbis nonstructural gene coding region terminatingin an inactivated junction region at Sindbis nt 7597, and 3′ viralelements necessary for genome replication; the firefly luciferase geneis placed between these two viral 5′ and 3′ elements. This vector isknown as pKSSINBVdlJR-luc.

The expression of the reporter gene from the pKSSINBVdlJR-luc vector istested in transfected BHK-21 cells. Translation of functional luciferaseprotein is determined by the luciferin luminescent assay, using aluminometer for detection. The sensitivity in this assay is 1×10⁻²⁰moles of luciferase. Given that the molecular weight of luciferase is62,000 daltons, this limit of detection transforms to 6,020 molecules.Thus, in a typical experiment if only 0.6% of the 1×10⁶ cells containedin a 60 mM petri dish are transfected with the pKSSINBVdlJR-luc vector,and if these transfected cells express only a single functional moleculeof luciferase, the enzymatic activity is detected by the assay used. Itis important to demonstrate in this experiment that the junction regionof the pKSSINBVdlJR-luc vector is inactivated. This is accomplished byan RNase protection assay, comparing the viral RNA's synthesized incells transfected with the pKSSINBVdlJR-luc and the pKSSINBV-lucvectors, using the probe described above.

The minimal −19→+5 junction region core oligonucleotide pair, comprisedof Sindbis nts 7579-7602is synthesized in vitro, and flanked with Apa Iand Xho I recognition sequences as shown:

oligonucleotide 1:

5′-CATCTCTACGGTGGTCCTAAATAGTC   (SEQ. ID NO. 26)

oligonucleotide 2:

5′-TCGAGACTATTTAGGACCACCGTAGAGATGGGCC   (SEQ. ID NO. 27)

The oligonucleotides above are mixed together in the presence of 10 mMMg²⁺, heated to 100° C. for 5 minutes and cooled slowly to roomtemperature. The annealed oligonucleotides are ligated at a 25:1 molarratio of insert to the pKSSINBVdlJR vector, prepared accordingly:complete digestion with Xho I, followed by digestion with Aga I underpartial conditions, resulting in one Apa I induced cleavage per molecule(of two cleavages possible), gel purification of the 10,655 bp fragment,and treatment with CIAP. This vector containing the entire nonstructuralprotein coding region which terminates in an inactivated junction regioncore, attached to a synthetic junction region core and followed by 3′viral elements required for replication, and contained in the pKSII+plasmid, is known pKSSINdlJRsjrc.

In order to regulate the level of subgenomic mRNA synthesis, furthermodifications of the tandemly inserted synthetic junction region core inplasmid pKSSINdlJRsjrc are performed. These modifications of thejunction region core may be accomplished by at least two approaches:nucleotide changes within the junction region core; or extension at the5′ and 3′ junction region core termini of flanking Sindbis nucleotides,according to the authentic viral sequence. The minimal junction regioncore, spanning viral nts 7579-7602 is shown below:

5′ATCTCTACGGTGGTCCTAAATAGT   (SEQ. ID NO. 2)

By comparing genomic sequence between eight alphaviruses, it has beenshown previously that there is sequence diversity within the junctionregion core. Shown below, for particular junction region locations, isthe Sindbis nucleotide followed by the corresponding nucleotide found inother alphaviruses:

Nucleotide Permissive Number Sindbis Change 7579 A C 7580 U C 7581 C U7583 C G 7589 U C 7590 G U 7591 G A 7592 U A 7600 A U or G 7602 U G or A

Junction region changes at Sindbis nts 7579, 7580, 7581, 7583, 7589,7590, 7591, 7592, result in potential amino acid coding changes withinall 5 condons of the carboxy terminus of NSP 4 which overlap in thejunction region. These changes observed in the junction region betweenalphaviruses at the level of NSP 4 coding potential and at the level ofjunction region cis activity may represent either, or both, permissivechanges in NSP 4 and the junction region which do not affectfunctionality, or on the other hand, simply different viruses. In anyevent, the junction region changes presented herein regard the tandemlyinserted junction region core, from which no NSP protein synthesisoccurs. Discussed above, translation of the entire NSP resion occursfrom the pKSSINBVdlJR construct. Junction region changes at Sindbis nts7600 and 7602 are downstream of the NSP 4 termination codon and upstreamof the structural proteins initiation codon.

Locations of nucleotide differences within the junction region coreobserved between the several alphavirus strains are referred to here aspermissive changes. Locations of nucleotides within the junction regioncore corresponding to conserved sequences between the several alphavirusstrains are referred to here as nonpermissive changes.

To decrease the level of subgenomic mRNA initiation from the syntheticjunction region core, changes are made separately within nucleotidescorresponding to permissive changes, and within nucleotidescorresponding to nonpermissive changes. Junction region nucleotidescorresponding to permissive changes are given in the table above.Fourteen junction region nucleotides for which no changes are observedamount the eight alphaviruses sequenced (Semliki Forest virus,Middleburg virus, Ross River virus, O'Nyong Nyong virus, Eastern EquineEncephalitis virus, Western Equine Encephalitis virus, and VenezuelanEquine Encephalitis virus) are given below:

Nucleotide Number:

7582

7584

7585

7586

7587

7588

7593

7594

7595

7596

7597

7598

7599

7601

Changes within the junction region observed among alphaviruses mayreflect a specific interaction between a given alphaviral RNA polymeraseand its cognate junction region. Thus, changes among the “permissive”nucleotides may result in as marked a decrease in the subgenomic mRNAsynthesis levels as changes amount the “nonpermissive” nucleotides ofthe junction region. On the other hand, these may indeed be sites ofpermissive change within the junction region core.

The single authentic nonpermissive change within the junction regioncore is likely Sindbis nt 7598, corresponding to the subgenomic mRNAinitiation point. Changes of this nucleotide in the tandemly insertedjunction region core of plasmid pKSSINdlJRsjrc are not described here.

Substitution of the permissive nucleotides in toto in the syntheticminimal −19→+5 junction region core, is accomplished with the followingoligonucleotide pair, synthesized in vitro, and flanked with Apa I andXho I recognition sequences as shown:

oligonucleotide 1:

5′-CCCTTGTACGGCTAACCTAAAGGAC   (SEQ. ID NO. 28)

oligonucleotide 2:

5′-TCGAGTCCTTTAGGTTAGCCGTACAAGGGGGCC   (SEQ. ID NO. 29)

The oligonucleotides above are mixed together in the presence of 10 mMMg, heated to 100° C. for 5 minutes and cooled slowly to roomtemperature. The annealed oligonucleotides are ligated at a 25:1 molarratio of insert to the pKSSINBVdlJR vector, prepared accordingly:complete digestion with Xho I, followed by digestion with Apa I underpartial conditions, resulting in one Apa I induced cleavage per molecule(of two cleavages possible), gel purification of the 10,655 bp fragment,and treatment with CIAP. This vector is known as pKSSINdlJRsjrPc.

Each of the 13 (nt 7598 not changed) nonpermissive nucleotides in thejunction region core are changed individually, using the followingrules, resulting in the most drastic transversional substitution:

A→C

T→G

G→T

C→A

For example, nt 7582 is changed from T→G, using the followingoligonucleotide pair, synthesized in vitro, and flanked with Apa I andXho I recognition sequences as shown:

oligonucleotide 1:

5′-CATCGCTACGGTGGTCCTAAATAGTC   (SEQ. ID NO. 30)

oligonucleotide 2:

5′-TCGAGACTATTTAGGACCACCGTAGCGATGGGCC   (SEQ. ID NO. 31)

(Nucleotides effecting transversion in nonpermissive junction regionsites shown in boldface type) The oligonucleotides above are mixedtogether in the presence of 10 mM Mg² 100° C. for 5 minutes and cooledslowly to room temperature. The annealed oligonucleotides are ligated ata 25:1 molar ratio of insert to the pKSSINBVdlJR vector, preparedaccordingly: complete digestion with Xho I, followed by digestion withApa I under partial conditions, resulting in one Apa I induced cleavageper molecule (of two cleavages possible), gel purification of the 10,655bp fragment, and treatment with CIAP. This vector is knownpKSSINdlJRsjrNP7582.

Using the transversion change rules shown above, changes in each of the12 remaining nonpermissive sites in the junction region core are madewith 12 separate oligonucleotide pairs, flanked with Apa I and Xho Irecognition sites, as described above. These vectors are known as:

pKSSINdlJRsjrNP7584

pKSSINdlJRsjrNP7585

pKSSINdlJRsjrNP7586

pKSSINdlJRsjrNP7587

pKSSINdlJRsjrNP7588

pKSSINdlJRsjrNP7593

pKSSINdlJRsjrNP7594

pKSSINdlJRsjrNP7595

pKSSINdlJRsjrNP7596

pKSSINdlJRsjrNP7597

pKSSINdlJRsjrNP7599

pKSSINdlJRsjrNP7601

In order to test the relative levels of subgenomic mRNA synthesis, theluciferase reporter gene is inserted into the modified tandem junctionregion vectors. This construction is accomplished by digesting with XhoI and Sac I and treating with CIAP the tandemly inserted syntheticjunction region core vectors and gel purifying the resulting approximate10,200 bp fragment. The treated vector fragment is then ligated togetherwith the 2854 bp small fragment resulting from digestion ofpKSII3′SIN-luc with Xho I and Sac I. These constructions contain theentire Sindbis nonstructural gene coding region terminating in aninactivated junction region at Sindbis nt 7597, the tandemly insertedsynthetic junction region core (modified or unmodified), the fireflyluciferase gene, and 3′ viral elements necessary for genome replication.The names of these vectors are as follows:

Tandemly Inserted Junction Region Sindbis-luciferase vector ModificationpKSSINd1JRsjrc-luc not modified pKSSINd1JRsjrPc-luc permissive changespKSSINd1JRsjrNP7582-luc nonpermissive change pKSSINd1JRsjrNP7584-lucnonpermissive change pKSSINd1JRsjrNP7585-luc nonpermissive changepKSSINd1JRsjrNP7586-luc nonpermissive change pKSSINd1JRsjrNP7587-lucnonpermissive change pKSSINd1JRsjrNP7588-luc nonpermissive changepKSSINd1JRsjrNP7593-luc nonpermissive change pKSSINdIJRsjrNP7594-lucnonpermissive change pKSSINd1JRsjrNP7595-luc nonpermissive changepKSSINd1JRsjrNP7596-luc nonpermissive change pKSSINd1JRsjrNP7597-lucnonpermissive change pKSSINd1JRsjrNP7599-luc nonpermissive changepKSSINd1JRsjrNP7601-luc nonpermissive change

Assuming that the translation efficiencies are equivalent in all of theluciferase vectors shown immediately above, the relative levels ofsubgenomic synthesis are determined by comparing the levels ofluciferase production at 16 hours post-transfection of BHK-21 cells. Therelative levels of subgenomic transcription are determined by comparingluciferase production by the vectors pKSSINBV-luc and pKSSINdlJRsjrc-lucwith all of the modified junction region luciferase vectors shown above.

Vectors containing the tandemly inserted synthetic junction region core(pKSSINdlJRsjrc, and derivatives thereof) should have a lower level ofsubgenomic mRNA expression, relative to the pKSSINBV construct.Therefore, in certain embodiments, it may be necessary to increase thelevel of subgenomic mRNA expression observed from the pKSSINdlJRsjrcvector. This may be accomplished by extension at the 5′ and 3′ syntheticjunction region core termini with 11 additional flanking Sindbisnucleotides, according to the authentic viral sequence.

The synthetic oligonucleotide pair shown below is synthesized in vitro,and contains 46 Sindbis nts, including all 24 nts (shown in boldfacetype) of the minimal junction region core. The Sindbis nts are flankedwith the Apa I and Xho I recognition sequences as shown:

oligonucleotide 1:

5′-CGGAAATAAAGCATCTCTACGGTGGTCCTAAATAGTCAGCATAGTACC   (SEQ. ID NO. 32)

oligonucleotide 2:

5′-TCGAGGTACTATGCTGACTATTTAGGACCACCGTAGAGATGCTTTA TTTCCGGGCC   (SEQ. IDNO. 33)

The oligonucleotides above are mixed together in the presence of 10 mMMg, heated to 100° C. for 5 minutes and cooled slowly to roomtemperature. The annealed oligonucleotides are ligated at a 25:1 molarratio of insert to the pKSSINBVdlJR vector, prepared accordingly:complete digestion with Xho I, followed by digestion with Apa I underpartial conditions, resulting in one Apa I induced cleavage per molecule(of two cleavages possible), gel purification of the 10,655 bp fragment,and treatment with CIAP. This vector containing the entire nonstructuralprotein coding region which terminates in an inactivated junction regioncore, attached to an extended synthetic junction region, and followed by3′ viral elements required for replication, and contained in the pKSII+plasmid, is known pKSSINjlJRsexjr.

In order to test the relative levels of subgenomic mRNA synthesis, theluciferase reporter gene is inserted into the extended tandem junctionregion pKSSINdlJRsexjr vector. This construction is accomplished bydigesting the pKSSINdlJRsexjr plasmid with Xho I and Sac I, treatingwith CIAP, and gel purifying the resulting approximate 10,200 bpfragment. The thus-treated vector fragment is ligated together with the2854 bp small fragment resulting from digestion of pKSII3′SIN-luc withXho I and Sac I. This construction contains the entire Sindbisnonstructural gene coding region terminating in an inactivated junctionregion at Sindbis nt 7597, the tandemly inserted extended syntheticjunction region, the firefly luciferase gene, and 3′ viral elementsnecessary for genome replication. The name of this vector ispKSSINjlJRsexjr-luc.

The relative levels of subgenomic transcription are determined bycomparing luciferase production by the pKSSINdlJRsexjr-luc vector withthe pKKINBV-luc and pKSSINdlJRsjrc-luc vectors.

E. CONSTRUCTION OF PLASMID DNA ALPHAVIRUS EXPRESSION VECTORS

The SINDBIS basic vector and SINDBIS-luciferase constructs described insections A and B of Example 3, above, are inserted into the pVGELVISvector configurations described previously in Example 2 such thatexpression of the heterologous gene from Sindbis vectors occurs afterdirect introduction of the plasmid DNA into cells. As described inExample 2, the ability to transfect alphavirus-based vector plasmid DNAdirectly onto cells resulting in expression levels of heterologous genestypical of transfection of RNA-based alphavirus vectors, without aprimary step consisting of in vitro transcription of linearized templatevector DNA, enhances greatly the utility and efficiency of certainembodiments of the alphavirus-based expression vector system. FIG. 8 isa schematic representation of one mechanism of expression ofheterologous genes from a plasmid DNA alphavirus expression (ELVIS)vectors. Primary transcription in the nucleus and transport of thevector RNA to the cytoplasm leads to the synthesis of alphavirusnonstructural proteins which catalyze the expansion of heterologous genemRNA via an antigenome intermediate which in turn serves as the templatefor production of genomic and subgenomic mRNA. The ELVIS vectors may beintroduced into the target cells directly by physical means as a DNAmolecule, as a complex with various liposome formulations, or as a DNAligand comples including the alphavirus DNA vector molecule, apolycation compound such as polylysine, a receptor specific ligand, and,optionally, a psoralen inactivated virus such as Sendai or Adenovirus.

The first step of constructing one representative plasmid DNA Sindbisexpression vector consists of digesting pKSSINBV with Sac I, bluntingwith T4 polymerase, digesting with Sfi I, isolating the 2,689 bpfragment, and ligating into the pVGELVIS 10,053 bp vector fragmentprepared by digestion with XbaI, blunting with T4 polymerase, digestingwith Sfi I, treatment with CIAP, and 1% agarose/TBE gel electrophoresis.This construction is known as pVGELVIS-SINBV.

In order to insert the luciferase gene into the pVGELVIS-SINBV vector,the SV40 intron and transcription termination sequences at the 3′-end ofluciferase must be removed so that when the pre-RNA, transcribed fromthe plasmid DNA luciferase vector after transfection into cells, isprocessed the 3′-end of the reporter gene is not separated from theSindbis vector 3′-end. The Sindbis 5′-and3′-ends contained within thepVGELVIS-SINBV vector are required in cis for the autocatalyticreplication activity of the vector. The Sindbis vector 3′-end isrequired for initiation of snythesis of the antigenomic strand, which isthe template for the subgenomic RNA encoding the heterologous orreporter protein.

The SV40 RNA processing signals positioned at the 3′-end of theluciferase gene are removed from the SIN-BV-luc construction describedin section B above. The modified luciferase fragment is then placed inthe pVGELVIS-SINBV construction described above via unique restrictionsties. The alteration of the luciferase gene is accomplished with theprimer pair shown below:

Forward primer 7328F (SIN nts 7328-7349):

5′-GTGGCGGATCCCCTGAAAAGG   (SEQ. ID NO. 10)

Reverse primer LucStop (buffer sequence/Not I, Xba I recognitionsequences/pGL-2 nts 1725-1703):

5′-TATATGCGGCCGCTCTAGATTACAATTTGGACTTTCCGCCC   (SEQ. ID NO. 34)

The primers shown above are used in a PCR reaction with a threetemperature cycling program using a 3 minute extension period. Theamplification products are purified with GENECLEAN®, digested with Xho Iand Xba I, purified again with GENECLEAN®, and the 2,037 bp fragment isligated into the 13,799 bp fragment of pVGELVIS-SINBV resulting fromdigestion with Xho I and Xba I, and treatment with CIAP. Thisconstruction is known as pVGELVIS-SINBV-luc (abbreviated as ELVIS-luc).

The expression of luciferase in BHK-21 cells transfected withpVGELVIS-SINBV-luc DNA is measured in order to demonstrate that theSindbis physical gene transfer vector is functional. Briefly, 5 ug ofpVGELVIS-SINBV-luc DNA or 5 ug of in vitro transcribed RNA fromlinearized SINBV-luc template as described in section B, above, arecomplexed with 10 ul of LIPOFECTAMINE® or LIPOFECTIN®, respectively, andtransfected into 5×10⁵ BHK-21 cells contained in 35 mM petri plates. Theluciferase activity is determined from each of three samples at 2, 4, 8,16, 20, 28, 48, 72, 96, and 120 hrs. post transfection. The results ofthis study, given in FIG. 9, demonstrate that the maximal level ofreporter gene expression from the pVGELVIS-SINBV-luc vector is similarto that observed in cells transfected with in vitro transcribed RNA fromlinearized SINBV-luc template. However, the luciferase activityexpressed from the pVGELVIS-SINBV-luc vector is at maximal levels atlater time points compared to that observed wit hthe SINBV-luc RNAvector, and continues at high levels while the activity from the RNAvector begins to diminish.

The following experiment is performed in order to demonstrate the levelof enhancement of heterologous gene expression provided by the ELVISvector system compared to the same RNA polymerase II promoter linkeddirectly to the luciferase gene reporter. Briefly, the Sindbis NSPs arefirst deleted from the pVGELVIS-SINBV-luc vector in order to demonstratethe requirement for the viral enzymatic proteins for high levels ofreporter gene expression. This is accomplished by digestion ofpVGELVIS-SINBV-luc DNA with Bsp EI, purification with GENECLEAN, andligation under dilute conditions. This construction is deleted ofnonstructural gene sequences between bases 422-7,054 and is analogous tothe pVGSP6GENdlBsp construction described in Example 3, section C aboveand shown schematically in FIG. 5. The construction described here isknown as pVGELVIS-SINBVdlBsp-luc (abbreviated as dlNSP ELVIS-luc). Tolink the luciferase gene directly to the MoMuLV LTR, the reporter isfirst inserted into the pCDNA3 vector (Invitrogen, San Diego, Calif.)between the Bam HI and Hind III sites. The luciferase fragment isderived from pGL2 plasmid exactly as described in Example 3 section B,above, and inserted into the 5428x bp fragment of pCDNA3 prepared bydigestion with Hind III and Bam HI, treatment with CIAP, andpurification on a 1% agarose/TBE gel. This construction is known aspCDNA3-luc. The U3 region of the MoMuLV LTR is amplified from the BAGvector using the PCR primers shown below as described in Example 2.

Forward primer: BAGBgl2F1 (buffer sequence/Bgl II recognitionsequence/Mo-MLV LTR nts 1-22):

5′-TATATAGATCTAATGAAAGACCCCACCTGTAGG   (SEQ. ID NO. 15)

Reverse primer: BAGwt441R2 (SIN nts 5-1/Mo-MLV LTR nts 441-406):

5′-TCAATCCCCGAGTGAGGGGTTGTGGGCTCTTTTATTGAGC   (SEQ. ID NO. 16)

The amplification products are purified with GENECLEAN and the ends arefirst blunted with T4 DNA polymerase, then digested with Bgl II,purified with GENECLEAN®′and ligated into the pCDNA3-luc plasmidprepared by digestion with Hind III, blunting with the Klenow enzyme and50 uM dNTPs, digestion with Bjl II, and purification by 1% agarose/TBEgel electrophoresis. This construction is known as LTR-luc.

The plasmids ELVIS-luc, dlNSP ELVIS-luc, LTR-luc, and ELVIS-luc dlproare each complexed with 10 ul of LIPOFECTAMINE® and transfected into5×10⁵ BHK-21 cells contained in 35 mM petri plates. The luciferaseactivity is determined from each of three samples at 48 hrs.post-transfection. The results of this study, given in FIG. 10,demonstrate that the level of heterologous gene expression enhancementprovided by the ELVIS system, compared to the same promoter linkeddirectly to the heterologous gene is at least 10-fold. The comparativelylow level of luciferase expression in cells transfected with the dlNSPELVIS-luc construction demonstrates that the expression enhancement is adirect result of functional Sindbis NSPs. The autocatalyticamplification of the reporter gene mRNA as depicted in FIG. 8 provides asignificant advantage in terms of levels of gene expression, compared toprimary transcription from simple promoter-heterologous geneconstructions. Thus, as shown schematically in FIG. 8, aftertransfection of the ELVIS vector primary transcription in the nucleusand transport of the vector RNA to the cytoplasm leads to the synthesisof Sindbis NSPs which catalyze the expansion of heterologous gene mRNAvia an antigenome intermediate which in turn serves as the template forproduction of genomic and subgenomic mRNA.

An experiment is performed to demonstrate the expression and rescue ofRNA- and plasmid DNA (ELVIS)-based Sindbis expression vectors. For theRNA vectors, 5×10⁵ BHK-21 cells contained in 35 mM petri plates aretransfected with SIN-luc RNA, or co-transfected with SIN-luc RNA andSINdlBspEI RNA, complexed with LIPOFECTIN®. For the ELVIS vectors, 5×10⁵BHK-21 cells contained in 35 mM petri plates are transfected withELVIS-luc, or co-transfected with ELVIS-luc and pVGELVISdlBspEI, whoseconstruction is described in Example 7, complexed with LIPOFECTAMINE®.The results of this study, shown in FIG. 23 demonstrate clearly that thelevel of expression after transfection and transduction is similarbetween BHK cells co-transfected with RNA or ELVIS vectors. Thus, theELVIS vectors are used not only as plasmid DNA expression vectors, butadditionally expression and helper vector ELVIS constructs can becotransfected into cells to generate recombinant vector particles.

F. CONSTRUCTION OF MODIFIED DNA-BASED ALPHAVIRUS EXPRESSION VECTORS

The overall efficiency of the ELVIS vector, as determined by level ofheterologous gene expression, is enhanced by several modifications tothe pVGELVIS-SINBV-luc vector. These modifications include alternate RNApolymerase II promoters and transcription termination signals, theaddition of intron sequences and ribozyme processing signals in thevector construct, and substitution with a smaller plasmid vectorbackbone. The construction of these modified ELVIS vectors is detailedbelow.

The modified ELVIS vector is assembled on the plasmid vector pBGS131(ATCC # 37443) which is a kanamycin resistant analogue of pUC9 (Sprattet al., Gene 41:337-342, 1986). Propagation of pBGS131 is in LB mediumwith 10 ug/ml kanamycin.

The transcription termination signals from the SV40 early region orBovine growth hormone are inserted between the Sac I and Eco RI sites ofpBGS131. The SV40 nts between viral nts 2643 to 2563 containing theearly region transcription termination sequences are isolated by PCRamplification using the primer pair shown below and the pBR322/SV40plasmid (ATCC # 45019) as template.

Forward primer SSVTT 2643 (buffer sequence/Sac I site/SV40 nts2643-2613):

5′-TATATATGAGCTCTTACAAATAAAGCAATAGCATCACAAATTTC   (SEQ. ID NO. 35)

Reverse primer RSVTT2563R (buffer sequence/Eco RI site/SV40 nts2563-2588):

5′TATATGAATTCGTTTGGACAAACCACAACTAGAATG   (SEQ. ID NO. 36)

The primers shown above are used in a PCR reaction with a threetemperature cycling program as described throughout this example, usinga 30 second extension period. The amplification products are purifiedwith GENECLEAN®, digested with Sac I and Eco RI, purified again withGENECLEAN®, and the 90 bp fragment is ligated into the 3,655 bp fragmentof pBGS131 resulting from digestion with Sac I and Eco RI, and treatmentwith CIAP. This construction is known as pBGS131-3′SV40TT

The Bovine growth hormone transcription termination sequences areisolated by PCR amplification using the primer pair shown below and thepCDNA3 plasmid (Invitrogen) as template.

Forward primer BGHTTF (buffer sequence/Sac I site/pCDNA3 nts 1132-1161):

5′-TATATATGAGCTCTAATAAAATGAGGAAATTGCATCGCATTGTC   (SEQ. ID NO. 37)

Reverse primer BGHTTR (buffer sequence/Eco RI site/pCDNA3 nts1180-1154):

5′-TATATGAATTCATAGAATGACACCTACTCAGACAATGCGATGC   (SEQ. ID NO. 38)

The primers shown above are used in a PCR reaction with a threetemperature cycling program, using a 30 sec. extension period. Theamplification products are purified with GENECLEAN®, digested with Sac Iand Eco RI, purified again with GENECLEAN®, and the 58 bp fragment isligated into the 3,655 bp fragment of pBGS131 resulting from digestionSac I and Eco RI, and treatment with CIAP. This construction is known aspBGS131-3′BGHTT.

In additional modifications to the ELVIS vector, the transcriptiontermination sequences are fused directly to the 3′-end Sindbissequences, resulting in deletion of the polyadenylate tract; oralternatively the antigenomic ribozyme sequence of hepatitis delta virus(HDV) is inserted between the 3′-polyadenylate tract of the ELVIS vectorand the transcription termination signals.

The HDV ribozyme-containing construct is generated with PCR techniquesand overlapping oligonucleotide primers which contain the minimal 84nucleotide antigenomic ribozyme sequence (Perotta and Been, Nature350:434-6, 1991). In addition to the HDV sequence, the primers containflanking Sac I recognition sites for insertion at the 3′ end of theELVIS vector. The HDV ribozyme sequence is generated with the threeoverlapping primers shown below.

Forward primer SHDV1F (Buffer sequence/Sac I site/HDV RBZ seq.):

5′-TATATGAGCTCGGGTCGGCATGGCATCTCCACCTCCTCGCGGTCCG   (SEQ. ID NO. 39)

Nested primer HDV17-68:

5′TCCACCTCCTCGCGGTCCGACCTGGGCATCCGAAGGAGGACGCAC GTCCACT-3′  (SEQ. ID NO.40)

Reverse primer SHDV84R (Buffer sequence/Sac I site/HDV RBZ seq.):

5′-TATATGAGCTCCTCCCTTAGCCATCCGAGTGGACGTGCGTCCTCCTTC   (SEQ. ID NO. 41)

The primers shown above are used in a PCR reaction with a threetemperature cycling program as described throughout this example, usinga 30 sec. extension period. The amplification products are purified withGENECLEAN®, digested with Sac I, purified again with GENECLEAN®, and the94 bp fragment subsequently is ligated into plasmid vectorspBGS131-3′SV40TT or pBGS131-3′BGHTT that are digested with SacI underlimiting conditions that linearize (cut 1 of 2 sites) and are treatedwith CIAP. These constructions are known as pBHGS131/HDV/3′SV40TT andpBGS131/HDV/3′BGHTT. Insertion of the HDV ribozyme in both the correctorientation and in the correct Sac I site is determined by sequencing.In addition, longer or shorter HDV ribozyme sequences, or any othercatalytic ribozyme sequence, may be readily substituted given thedisclosure provided herein.

In the second vector 3′-end configuration, the SV40 or BGH transcriptiontermination signals are fused directly to the 3′-end of the ELVIS vectorcorresponding to Sindbis nt 11,700 and the polyadenylate tract isdeleted. This construction is accomplished according to the stepsoutlined above in Example 3, sections A and B for the assembly of thepKSSINBV and pKSSINBV-luc vectors. However, in this application thevector 3′-end primer does not contain a 25 polyadenylate tract. The3′-end of the vector is synthesized with the primer pair shown below:

Forward Primer: SIN11664F: (buffer sequence/Not I site/SIN nts11664-11698):

5′-TATATGCGGCCGCTTTCTTTTATTAATCAACAAAATTTTGTTTTTAA   (SEQ. ID NO. 42)

Reverse Primer: SSIN11700R (buffer sequence/Sac I site/SIN nts11700-11655:

5′-TATATGAGCTCGAAATGTTAAAAACAAAATTTTGTTG   (SEQ. ID NO. 43)

The primers shown above are used in a PCR reaction with a threetemperature cycling program as described throughout this example, usinga 30 sec. extension period. Assembly of the pKSSINBV and pKSSINBV-lucvectors is precisely as shown in Example 3, sections A and B. Theseconstructions are known as pKSSINBVdlA and pKSSINBVdlA-luc.

The ELVIS expression vectors are assembled further onto the various 3′end processing plasmid constructions described above. The Sindbisvectors containing a polyadenylate tract are combined with the plasmidconstructions containing the HDV ribozyme sequence and the SV40 BGHtranscription termination signals. This construction corresponds to theinsertion of pKSSINBV and pKSSINBV-luc vector sequences into thepBGS131/HDV/3′SV40TT and pBGS131/HDV/3′BGHTT plasmids. Alternatively,the Sindbis vectors terminating precisely at the viral 3′ endcorresponding to viral nt 11,700 are linked directly to the SV40 or BGHtranscription termination signals. This construction corresponds to theinsertion of pKSSINBVdlA and pKSSINBVdlA-luc vector sequences into thepBGS131/HDV/3′SV40TT and pBGS131/HDV/3′BGHTT plasmids.

The Sindbis vectors pKSSINBV and pKSSINBV-luc are digested with Sac Iand Bgl II, and the 5,222 bp (pKSSINBV) or 8211 bp (pKSSINBV-luc)fragments are purified by 1% agarose/TBE gel electrophoresis andinserted into the linearized pBGS131/HDV/3′SV40TT andpBGS131/HDV/3′BGHTT plasmids prepared by digestion with Sac I and Bgl IIand treatment with CIAP. These constructions are known as:

pBGS131/dlproSINBV-luc/HDV/3′SV40TT

pBGS131/dlproSINBV-luc/HDV/3′BGHTT

pBGS131/dlproSINBV/HDV/3′SV40TT

pBGS131/dlproSINBV/HDV/3′BGHTT

Using the same strategy described above, the Sindbis vectors pKSSINBVdlAand pKSSINBVdlA-luc are digested with Sac I and Bgl II, and the 5,497 bp(pKSSINBVdlA) or 8,186 bp (pKSSINBVdlA-luc) fragments are purified by 1%agarose/TBE gel electrophoresis and inserted into the linearizedpBGS131/3′SV40TT and pBGS131/3′BGHTT plasmids prepared by digestion withSac I and Bgl II and treatment with CIAP. These constructions are knownas:

pBGS131/dlproSINBV-luc/3′SV40TT

pBGS131/dlproSINBV-luc/3′BGHTT

pBGS131/dlproSINBV/3′SV40TT

pBGS131/dlproSINBV/3′BGHTT

The addition of an RNA polymerase II promoter and Sindbis nucleotides1-2289 is the last step required to complete the construction of themodified ELVIS expression vectors of the eight constructions shownbelow:

pBGS131/dlproSINBV-luc/HDV/3′SV40TT

pBGS131/dlproSINBV-luc/HDV/3′BGHTT

pBGS131/dlproSINBV/HDV/3′SV40TT

pBGS131/dlproSINBV/HDV/3′BGHTT

pBGS131/dlproSINBV-luc/3′SV40TT

pBGS131/dlproSINBV-luc/3′BGHTT

pBGS131/dlproSINBV/3′SV40TT

pBGS131/dlproSINBV/3′BGHTT

These eight constructions contain a unique Bgl II restriction site,corresponding to Sindbis nt 2289. The RNA polymerase II promoter andSindbis nucleotides 1-2289 are inserted into these constructions by theoverlapping PCR technique described for the pVGELVIS construction inExample 2. In order to insert the RNA polymerase II promoter and the2289 Sindbis nts, the eight constructions shown above are digested withBgl II and treated with CIAP.

The U3 region of the long terminal repeat (LTR) from Moloney murineleukemia virus (Mo-MLV) is positioned at the 5′ viral end such that thefirst transcribed nucleotide is a single G residue, which is capped invivo, followed by the Sindbis 5′ end. Amplification of the Mo-MLV LTR inthe first primary PCR reaction is accomplished in a reaction containingthe BAG vector (Price et al., PNAS 84:156-160, 1987) and the followingprimer pair:

Forward primer: BAGBgl2F1 (buffer sequence/Bgl II recognitionsequence/Mo-MLV LTR nts 1-22):

5′-TATATAGATCTAATGAAAGACCCCACCTGTAGG   (SEQ. ID NO. 15)

Reverse primer: BAGwt441R2 (SIN nts 5-1/Mo-MLV LTR nts 441-406):

5′-TCAATCCCCGAGTGAGGGGTTGTGGGCTCTTTTATTGAGC   (SEQ. ID NO. 16)

The primers shown above are used in a PCR reaction with a threetemperature cycling program using a 30 second extension period.

Amplification of the Sindbis 5′ end in the second primary PCR reactionis accomplished in a reaction containing the pVGSP6GENrep clone and thefollowing primer pair:

Forward primer: (Mo-MLV LTR nts 421-441/SIN nts 1-16):

5′-CCACAACCCCTCACTCGGGGATTGACGGCGTAGTAC   (SEQ. ID NO. 17)

Reverse primer: (SIN nts 3182-3160):

5′-CTGGCAACCGGTAAGTACGATAC   (SEQ. ID NO. 18)

The primers shown above are used in a PCR reaction with a threetemperature cycling program using a 3 minute extension period.

The 457 bp and 3202 bp products from the primary PCR reactions arepurified with GENECLEAN™, and used together in a PCR reaction with thefollowing primer pair:

Forward primer: BAGBgl2F1 (buffer sequence/Bgl II recognitionsequence/Mo-MLV LTR nts 1-22):

5′-TATATAGATCTAATGAAAGACCCCACCTGTAGG   (SEQ. ID NO. 15)

Reverse primer: (SIN nts 2300-2278):

5′-GGTAACAAGATCTCGTGCCGTG   (SEQ. ID NO. 19)

The primers shown above are used in a PCR reaction with a threetemperature cycling program using a 3 minute extension period. The 253′-terminal bases of the first primary PCR amplicon product overlap withthe 25 5′-terminal bases of the second primary PCR amplicon product; theresultant 2,752 bp overlapping secondary PCR amplicon product ispurified by 1% agarose/TBE electrophoresis, digested with Bgl II, andthe 2,734 bp product is ligated into the eight ELVIS constructionsdescribed above. These constructions are named as shown below:

MpLTRELVIS-luc/D/S

MpLTRELVIS-luc/D/B

MpLTRELVIS/D/S

MpLTRELVIS/D/B

MpLTRELVIS-luc/S

MpLTRELVIS-luc/B

MpLTRELVIS/S

MpLTRELVIS/B

Using the same overlapping PCR approach, the CMV promoter is positionedat the 5′ viral end such that transcription initiation results in theaddition of a single non-viral nucleotide at the Sindbis 5′ end.Amplification of the CMV promoter in the first primary PCR reaction isaccomplished in a reaction containing the pCDNA3 plasmid and thefollowing primer pair:

Forward primer: pCBgl/233F (buffer sequence/Bgl II recognitionsequence/CMV promoter nts 1-22):

5′-TATATATAGATCTTTGACATTGATTATTGACTAG   (SEQ. ID. NO. 44)

Reverse primer: SNCMV1142R (SIN nts 8-1/CMV pro nts 1142-1108):

5′-CCGTCAATACGGTTCACTAAACGAGCTCTGCTTATATAGACC   (SEQ. ID NO. 45).

The primers shown above are used in a PCR reaction with a threetemperature cycling program using a 1 minute extension period.

Amplification of the Sindbis 5′ end in the second primary PCR reactionis accomplished in a reaction containing the pVGSP6GENrep clone and thefollowing primer pair:

Forward primer: CMVSIN1F (CMV pro nts 1124-1142/SIN nts 1-20):

5′-GCTCGTTTAGTGAACCGTATTGACGGCGTAGTACACAC   (SEQ. ID NO. 46).

Reverse primer: (SIN nts 3182-3160):

5′-CTGGCAACCGGTAAGTACGATAC   (SEQ. ID NO. 18).

The primers shown above are used in a PCR reaction with a threetemperature cycling program using a 3 minute extension period.

The 600 bp and 3200 bp products from the primary PCR reactions arepurified with GENECLEAN™, and used together in a PCR reaction with thefollowing primer pair:

Forward primer: pCBgl233F (buffer sequence/Bgl II recognitionsequence/CMV promoter nts 1-22):

5′-TATATATAGATCTTTGACATTGATTATTGACTAG   (SEQ. ID NO. 44).

Reverse primer: (SIN nts 2300-2278):

5′-GGTAACAAGATCTCGTGCCGTG   (SEQ. ID. NO. 19)

The primers shown above are used in a PCR reaction with a threetemperature cycling program using a 3 minute extension period.

The 26 3′ terminal bases of the first primary PCR amplicon productoverlaps with the 26 5′ terminal bases of the second primary PCRamplicon product; the resultant 2,875 bp overlapping secondary PCRamplicon product is purified by 1% agarose/TBE electrophoresis, digestedwith Bgl II, and ligated into the four ELVIS constructions describedabove. These constructions are named as shown below:

MpCMVELVIS-luc/D/S

MpCMVELVIS-luc/D/B

MpCMVELVIS/D/S

MpCMVELVIS/D/B

MpCMVELVIS-luc/S

MpCMVELVIS-luc/B

MpCMVELVIS/S

MpCMVELVIS/B

Using the same overlapping PCR approach, the SV40 early region promoteris positioned at the 5′ viral end such that the major cap site oftranscription initiation results in the addition of a single non-viralnucleotide at the Sindbis 5′ end. Amplification of the SV40 promoter inthe first primary PCR reaction is accomplished in a reaction containingthe pBR322/SV40 plasmid (ATCC #45019) and the following primer pair:

Forward primer: B2SVpr250F (buffer sequence/Bgl II recognitionsequence/SV40 nts 250-231):

5′-TATATATAGATCTGGTGTGGAAAGTCCCCAGGC   (SEQ. ID NO. 47)

Reverse primer: SINSV5235R (SIN nts 13-1/SV40 nts 5235-10):

5′-CTACGCCGTCAATGCCGAGGCGGCCTCGGCC   (SEQ. ID NO. 48)

The primers shown above are used in a PCR reaction with a threetemperature cycling program using a 30 second extension period.

Amplification of the Sindbis 5′ end in the second primary PCR reactionis accomplished in a reaction containing the pVGSP6GENrep clone and thefollowing primer pair:

Forward primer: SVSIN1F (SV40 nts 3-5235/SIN nts 1-25):

5′-GGCCGCCTCGGCATTGACGGCGTAGTACACACTATTG   (SEQ. ID NO. 49)

Reverse primer: (SIN nts 3182-3160):

5′-CTGGCAACCGGTAAGTACGATAC   (SEQ. ID NO. 18)

The primers shown above are used in a PCR reaction with a threetemperature cycling program using a 3 minute extension period.

The 280 bp and 3,194 bp products from the primary PCR reactions arepurified with GENECLEAN™, and used together in a PCR reaction with thefollowing primer pair:

Forward primer: B2SVpr250F (buffer sequence/Bgl II recognitionsequence/SV40 nts 250-231):

5′-TATATATAGATCTGGTGTGGAAAGTCCCCAGGC   (SEQ. ID NO. 47)

Reverse primer: (SIN nts 2300-2278):

5′-GGTAACAAGATCTCGTGCCGTG   (SEQ. ID NO. 19)

The primers shown above are used in a PCR reaction with a threetemperature cycling program using a 3 minute extension period.

The 25 3′ terminal bases of the first primary PCR amplicon productoverlaps with the 25 5′ terminal bases of the second primary PCRamplicon product; the resultant 2,543 bp overlapping secondary PCRamplicon product is purified by 1% agarose/TBE electrophoresis, digestedwith Bgl II, and ligated into the four ELVIS constructions describedabove. These constructions are named as shown below:

MpSV40ELVIS-luc/D/S

MpSV40ELVIS-luc/D/B

MpSV40ELVIS/D/S

MpSV40ELVIS/D/B

MpSV40ELVIS-luc/S

MpSV40ELVIS-luc/B

MpSV40ELVIS/S

MpSV40ELVIS/B

The luciferase expression levels, after transfection of BHK-21 cells,are determined with each of the reporter gene containing completemodified ELVIS constructions detailed above, in order to determine theoptimal desired configuration. The heterologous gene is inserted intothe multiple cloning site of the ELVIS vector, as described for theinsertion of the luciferase gene in Example 3, section B.

In order to increase the efficiency of the ELVIS system, in terms offunctional vector RNA transported to the cytoplasm per nuclear DNAtemplate, the SV40 small t antigen intron can be inserted into the ELVISexpression vectors. Insertion of the SV40 small t antigen intronsequences into the Xho I site immediately downstream of the 5′ Sindbissequences is accomplished by limited digestion (cut 1 and 2 sites); or,alternatively at the unique Not I site immediately upstream of the 3′Sindbis sequences.

For insertion into the Xho I site of the ELVIS vectors, amplification ofthe SV40 small t antigen intron sequences is accomplished in a reactioncontaining the pBR322/SV40 plasmid (ATCC #45019) and the followingprimer pair:

Forward primer: XSVSD4647F (buffer sequence/Xho I recognitionsequence/SV40 nts 4647-4675):

5′-TATATATCTCGAGAAGCTCTAAGGTAAATATAAAATTTACC   (SEQ. ID NO. 50)

Reverse primer: XSVSA4562R (buffer sequence/Xho I recognitionsequence/SV40 nts 4562-4537):

5′-TATATATCTCGAGAGGTTGGAATCTAAAATACACAAAC   (SEQ. ID NO. 51)

The primers shown above are used in a PCR reaction with a threetemperature cycling program using a 30 second extension period. Theamplification products are purified with GENECLEAN™, digested with XhoI, re-purified with GENECLEAN™ and inserted into Xho I linearized (bylimited digest) and CIAP treated complete modified ELVIS vectorsdescribed above. Insertion of the SV40 small t antigen intron in thecorrect orientation in the ELVIS vector is determined by sequencing.

For insertion into the Not I site of the ELVIS vectors, amplification ofthe SV40 small t antigen intron sequences is accomplished in a reactioncontaining the pBR322/SV40 plasmid and the following primer pair:

Forward primer: NSVSD464F (buffer sequence/Not I recognitionsequence/SV40 nts 4647-4675):

5′-TATATATGCGGCCGCAAGCTCTAAGGTAAATATAAAATTTACC   (SEQ. ID NO. 52)

Reverser primer: XSVSA4562R (buffer sequence/Not I recognitionsequence/SV40 nts 4562-4537):

5′-TATATATGCGGCCGCAGGTTGGAATCTAAAATACACAAAC   (SEQ. ID NO. 53)

The primers shown above are used in a PCR reaction with a threetemperature cycling program using a 30 second extension period. Theamplification products are purified with GENECLEAN™, digested with NotI, re-purified with GENECLEAN™ and inserted into Not I linearized andCIAP treated complete modified ELVIS vectors described above. Insertionof the SV40 small t antigen intron in the correct orientation in theELVIS vector is determined by sequencing. Alternatively, the SV40 smallt antigen may be inserted at other sites within the ELVIS vector, whichdo not impair function of the vector, using the disclosure providedherein.

The luciferase expression levels, after transfection of BHK-21 cellswith the SV40 small t antigen intron containing ELVIS vectors, areassayed in order to determine the optimal desired configuration. Theheterologous gene is inserted into the multiple cloning site of theELVIS vector, as described for the insertion of the luciferase gene inExample 3, section B.

A linker sequence is inserted into the pKSSINBV and into the pVGELVIS-SINBV constructs to facilitate the insertion of heterologoussequences. The liner is constructed using two complementary 35ntoligonucleotides that form a duplex with Xho I and Xba I compatiblesticky ends when hybridized.

SINBVLinkF: 5′TCGAGCACGTGGCGCGCCTGATCACGCGTAGGCCT   (SEQ. ID NO. 54)

SINBVLinkR: 5′CTAGAGGCCTACGCGTGATCAGGCGCGCCACGTGC   (SEQ. ID NO. 55)

The oligonucleotides are phosphorylated with T4 polynucleotide kinase,heated to 90° C., and slow cooled to allow hybridization to occur. Thehybrid is then ligated to the 10.6kb fragment of pKSSINBV-Luc obtainedafter digestion with XhoI and XbaI, followed by treatment with alkalinephosphatase and agarose gel purification. The resulting constructcontains Xho I, Pml I, Asc I, Bcl I, Mlu I, Stu I, Xba I, and Not I asunique sites between the Sindbis junction region and the Sindbis 3′ end.This construct is known as pKSSINBV-Linker.

This linker also is cloned into the pVGELVIS-SINBV constructs. Thelinker is inserted by digestion of pVGELVIS-SINBV-luc with Sfi I and NotI. The 10kb fragment is agarose gel purified, and this fragment wasligated to the gel purified 2.6kb fragment from a SfiI/Not I digest ofpKSSINBV-Linker. The resulting construct contains Xho I, Pml I, Asc I,Mlu I, and Not I as unique sites between the Sindbis junction region andthe Sindbis 3′ end. This construct is known as pVGELVIS-SINBV-Linker.

Additional experiments are performed to compare the relative expressionactivities of Sindbis RNA and DNA reporter vectors in transfected BHKcells (FIG. 22). Luciferase expression is approximately 30-fold higherin cells transfected with in vitro transcribed SIN-luc RNA, compared tothe level in cells transfected with ELVIS-luc plasmid DNA (FIG. 22A).The data also demonstrate that direct linkage between the Sindbis virus3′-end and two different transcription termination/polyadenylationsignals, resulting in deletion of the synthetic A25tract, decreased theactivity of the DNA vector by more than three orders of magnitude (FIG.22A). However, measurable expression of luciferase is detected,suggesting that these 3′ end modified Sindbis DNA vectors do function intransfected cells at some low level. Additionally, the insertion of aHDV ribozyme processing sequence, downstream of the A25 tract, increasesactivity of the DNA vector 3-4 fold over the ELVIS-luc vector or ananalogous construct with the HDV ribozyme inserted in a reverseorientation (FIG. 22B).

Based on the decreased expression levels observed when the synthetic A25tract is deleted, additional constructs related to MpELVIS/S andMpELVIS/B are then made exactly as outlined in the above exampleutilizing the Sindbis sequences from the pKSSINBV and pKSSINBV-lucvectors to include the A25 tract. These constructions are named as shownbelow:

MpLTRELVIS-luc/A/S

MpLTRELVIS-luc/A/B

MpLTRELVIS/A/S

MpLTRELVIS/A/B

MpCMVELVIS-luc/A/S

MpCMVELVIS-luc/A/B

MpCMVELVIS/A/S

MpCMELVIS/A/B

MpSV40ELVIS-luc/A/S

MpSV40ELVIS-luc/A/B

MpSV40ELVIS/A/S

MpSV40ELVIS/A/B

G. REPORTER GENE EXPRESSION IN RODENTS INOCULATED INTRAMUSCULARLY WITHELVIS VECTORS

Using techniques described above, the lacZ gene encoding the β-galactosidase reporter protein was cut from the plasmidpSV-β-galactosidase (PROMEGA CORP, Madison, Wis.) and substituted intothe ELVIS-luc plasmid DNA vector in place of luciferase. To examine invivo gene expression from ELVIS vectors, Balb/c mice and rats areinjected intramuscularly (i.m.) with ELVIS-β-gal or ELVIS-luc plasmidDNA vectors. FIG. 24 demonstrates the in vivo expression ofβ-galactocidase in muscle tissue taken from a rat and stained with X-galat three days post i.m. injection. Mice injected with ELVIS-β-gal alsodemonstrate positively staining blue muscle fibers. Luciferaseexpression levels from muscle which were between 75- and 300-fold higherthan control levels were detected in ¾ Balb/c mice at two days post i.m.inoculation with ELVIS-luc plasmid. In other experiments, C3H/HeN micewere injected i.m. with ELVIS vectors expressing either the hepatitis Bvirus core (HBV-core) or hepatitis B virus e (HBV-e) proteins. UsingELISA detection systems, both HBV-core- and HBV-e-specific IgGantibodies were detected in serum samples collected from the mice 10days following the second injection with the vectors. These experimentsdemonstrate that Sindbis-derived DNA vectors are able to express foreigngenes in vivo, in rare and mouse muscle.

H. ADAPTATION OF ALPHAVIRUS EXPRESSION VECTORS

The following description details how to identify alphaviral vectorsaccording to the invention adapted to grow in cells of a particulareukaryotic species. Specifically, adaptation of Sindbis virus variantsadapted to grow in human cells is disclosed. As those in the art willappreciate, the following procedure can be employed to adapt otheralphaviral vectors to particular eukaryotic species.

To adapt Sindbis viral vectors derived from BHK-21 cells to human cellsSindbis viral vectors produced in accordance with this invention arepropagated by serial passage in HT1080 (ATCC acc. no. CCL 121) and DM150(a human cell line established from a primary melanoma tumor) cell linesin order to select variants which are able to establish high titerproductive infections in human cells. Isolation of Sindbis variantsadapted to human cell is accomplished by the following method: HT1080and DM150 cells propagated in DMEM with 10% fetal calf serum (FCS) areinfected at a multiplicity of infection of 5 with the virus contained ina small volume to facilitate infection. At one hour post infection, theinoculum is removed, the monolayer washed several times with DMEM, andthe media replenished. The viral supernatant is harvested at 7 hourspost infection, clarified by centrifugation, and divided into threealiquots. Two aliquots are frozen and the other aliquot is split andused to infect fresh HT1080 and DM150 monolayers. This process isrepeated at least 10 times or as sufficient to generate variants whichreplicate efficiently in human cells. After each serial passage, plaqueassays are performed in BHK cells or the homologous cell line in whichthe virus was propagated to determine an increase in virus titer inhuman cell lines. Sindbis variants adapted to human cells which containthe highest level of virus produced during serial HT1080 or DM150 cellline passage are then isolated from supernatants by three rounds ofplaque purification. The phenotype of the plaque purified human variantis verified by determining its growth properties in human cell lines.

In an alternative approach, variants which are able to establish hightiter productive infections in human cells are isolated by plaquemorphology. Human cell lines, for example HT1080 and DM150 , areinfected at low multiplicity of infection with Sindbis virus grown inBHK-21 cells and overlaid with agar. At 24-30 hours post infection,large plaques, indicative of variants able to propagate efficiently inhuman cells, are picked. The variants are then purified by twoadditional serial rounds of plaque purification. The phenotype ofcandidate Sindbis variants can then be determined by comparing growthproperties on human and BHK-21 cells with BHK-21 cell-propagated Sindbisvirus.

Another similar approach enables the production of Sindbis variantswhich establish high titer persistent, i.e., noncytotoxic, infection ofhuman cells. Specifically, human cells are infected with a Sindbis viruspreparation containing a high percentage of defective interfering (DI)particles isolated by undiluted serial passage in HT1080 or DM150 cells.Cells which survive infection with this DI contaminated Sindbis stockare allowed to proliferate. Virus is isolated from the supernatant andpurified by multiple rounds of plaque purification in BHK-21 or humancells. The desired phenotype of the Sindbis variant is verified bydetermining its ability to establish persistent noncytotoxic persistentinfection in human cell lines.

Following identification of one or more Sindbis variants having thedesired phenotype, purified viral RNA from the Sindbis variant is clonedand characterized in order to identify the nonstructural and structuralgenes and noncoding region changes which contribute to the observedphenotype. Sindbis variant genomic cDNA cloning is accomplished byRT-PCR, as detailed in Example 1 and the phenotype of the molecularlycloned virus strains is verified.

Viral genetic determinants can be mapped by identifying at what levelSindbis infection of human cells is inhibited, i.e., at the stage ofadsorption, entry, replication, or assembly. The 5′-end, junctionregion, and nonstructural and noncoding region genetic determinantsresponsible for human variant phenotypes can be mapped by exchangingdefined regions from pKSSINBV-luc, supra, with corresponding regionsfrom the variant cDNA to product various “test” SIN-luc vectors. Afterpackaging by co-transfection, the level of luciferase expression inDM150, HT1080, and BHK cells infected with either pKSSINBV-luc or the“test” SIN-luc vector is compared. Exchanging defined regions betweenvectors may be accomplished by exploiting convenient restrictionendonuclease recognition sites, for example (Viagene SIN-BV numbering):Afl II (4573), Age I (3712, 6922), Avr II (4281), Bgl II (2289),Bpu1102I (5602, 6266), BsaBI (2479) BstBI (4706, 6450), Eco47III (1407),Hpa I (6920), Mun I (42, 2785), Nru I (2324), Nsi I (2006, 6462), PflMI(4374), Sfi I (5122), and XhoI (7645). Precise nucleotide identificationof genetic determinants resulting in the human variant phenotype can beaccomplished by sequencing.

The 3′-end nonstructural and coding region genetic determinantsresponsible for the variant phenotype may be mapped by exchangingdefined regions with the dl-BspEI cotransfection packaging vector. Afterpackaging by co-transfection, the level of luciferase expression inDM150, HT1080, and BHK cells infected with pKSSINBV-luc packaged withthe dl-BspEI cotransfection packaging vector or with the “test ”dl-BspEI cotransfection packaging vector is compared. Exchanging definedregions between vectors may be accomplished by exploiting convenientrestriction endonuclease recognition sites, for example (Viagene genomicSindbis numbering): AatII (8000), Afl II (7969, 8836), AvaI (9414), BelI(9356), Bpu 1102I (8911), BsiWI (10379), BspMII (7054), Bsu36I (8892),EcoNI (10048, 10923), EcoRI (9077), KasI (10036, 11308), NruI (8329),PflMI (9554), PmlI (8070), SalI (9589, 11085), SmalI (9416),SplI(10379), StuI (8572), and (9414). Precise nucleotide identificationof genetic determinants resulting in the human variant phenotype can beaccomplished by sequencing.

I. RECOMBINANT PROTEIN EXPRESSION

The eukaryotic layered vector initiation systems of the invention can beused to direct the expression of one or more recombinant proteins intransformed or transfected eukaryotic host cells. A representativeexample of a recombinant protein which may be expressed using aeukaryotic layered vector initiation system is insulin.

The gene encoding human insulin was identified in 1980 by Bell, et al.,[Nature, vol. 284, pp. 26-32]. The entire coding region for humanpreproinsulin (hppi) can be cloned from a variety of sources, e.g., ahuman pancreatic cDNA library [Clontech, Palo Alto, Calif., catalog no.HL1163a] using standard PCR techniques. Primers for amplifying thecoding region flank the 5′ and 3′ ends of the gene. The 5′ primerincludes an XhoI site and the 3′ primer includes a NotI recognitionsequence. After PCR amplification, the reaction products are purifiedusing GENECLEAN™, followed by XhoI and NotI digestion. The DNA is thengel purified and ligated into XhoI/NotI cleaved, CIAP-treatedpVGELVIS-SINBV, infra, to make pELVS-hppi.

Alternatively, the hppI amplicon is inserted into XhoI/Not I cleaved,CIAP-treated SIN-BV, infra, to make pSIN-BV-hppI. RNA from SacI-linearized pSIN-BV-hppI plasmid is synthesized in vitro as describedin Example 3. Production of SIN-BV-hppI recombinant vector particles isaccomplished by transfection of LIPOFECTIN™-complex SIN-BV-hppI RNA intothe Sindbis vector packaging cell lines as described in Example 7.Generation of vector particles having expression vectors derived fromSindbis variants which establish high titer persistent noncytotoxicinfection of human cells is accomplished by the same procedure.

pELVS-hppi is then introduced (e.g., by electroporation or by complexingwith lipofectamine) into a suitable eukaryotic host cell, preferably anundifferentiated cell, for instance, F9 cells, infra. The transformedcells are then grown in the presence of G418 under suitable nutrientconditions (i.e., an appropriate medium, such as DMEM, including anyrequired supplements, at 37° C.). The cells can be grown in a variety offormats, including in roller bottles, cell hotels, and bioreactors.Recombinant protein production is initiated by adding retinoic acid oranother suitable inducing agent to the medium. At 12 to 48 hourspost-vector induction, the optimal level of insulin is expressed intothe medium and is recovered according to techniques known in the art.The insulin is recovered from the cell supernatants up to 18 hrs fromthe time in which the vector establishes a cytotoxic infection. Recoveryof insulin from cells infected with expression vectors derived fromSindbis human cell variants may be harvested over a period extending to3-5 days post induction. Insulin so produced is recovered according totechniques known in the art. The isolated recombinant protein may thenbe formulated in any of a number of pharmaceutical compositions suitablefor human administration.

J. LYOPHILIZED EUKARYOTIC LAYERED VECTOR INITIATION SYSTEM VACCINES

One aspect of the invention concerns the use of eukaryotic layeredvector initiation systems according to the invention as vaccines toimmunize a human patient's or non-human animal's immune system against aparticular disease. Such vaccines can be employed either prophylaticallyor therapeutically to prevent or treat disease. Diseases which may betreated with such vaccines include those caused by various pathogenicagents, such as procaryotic or eukaryotic microorganisms or viruses, orcancer.

For example, each of the vector constructs described herein andcontaining the heterologous sequence of a suitable antigen is readilylyophilized for long term stability. Upon re-hydration in an appropriatediluent, administration is performed and subsequent expression occurs.Additional alphavirus vector constructs not disclosed in the presentinvention, including those described in the literature (see Hahn et al.,Proc Natl Acad Sci USA 89: 2679-2683, 1992) are readily convertible to aeukaryotic layered vector initiation system format by those skilled inthe art and using the knowledge provided herein. Conversion of transientalphavirus vector systems to the format of a eukaryotic layered vectorinitiation system thus modify the duration of heterologous sequenceexpression to that of a more permanent and stable expression system.Advantages of this permanent and stable system include longer termexpression, allowing greater prophylatic and therapeutic effects in bothmedical and veterinary applications.

K. EUKARYOTIC LAYERED VECTOR INITIATION SYSTEMS FOR PLANT APPLICATIONS

Given the disclosures provided herein, the adaptation of eukaryoticvector initiation system technologies to plant application is readilyperformed by those skilled in the art. For illustration purposes, any ofseveral positive-stranded plant viruses (for example, potato virus X(PVX, Huisman et al., J. Gen. Virol. 69:1789-1798, 1988) tobacco mosaicvirus (TMV, Goelet et al., Proc. Natl. Acad. Sci USA 79:5818-5822,1982), and tobacco etch virus (TEV, Allison et al., Virology 154:9-20,1986), see also, specifications) may be converted to a cDNA form usingPCR and specific oligonucleotide primers, chosen from publishedsequences, as described in Example 1. After assembly of a full-lengthgenomic clone linked to a bacteriophage RNA polymerase promoter, anddetermination of infectivity of in vitro synthesized transcripts, thecDNA is exchanged into a vector containing an RNA polymerase II promoterand transcription termination/polyadenylation sequence, as described inExample 2. For plant applications, such promoter and terminationsequences are chosen from the appropriate plant systems (e.g., CaMV 35Spromoter (Guilley et al., Cell 30:763-773, 1982,), and nopaline synthasepromoter and transcription termination sequence (Sanders et al., NucleicAcids Res. 15:1543-1558). Vector constructs derived from theseinfectious genomic cDNA clones is subsequently accomplished using any ofthe approaches described in the present invention (e.g., use ofsubgenomic promoters, replacement of structural protein genes, use inIRES sequences). Specific applications of such plant eukaryotic layeredvector initiation systems may include, but are not limited to, theexpression of host-derived resistance sequences, pathogen-derivedresistance sequences (e.g., protein-encoding, nonprotein-encoding, anddefective interfering sequences), and growth promoting sequences, by thecreation of transgenic plants harboring such systems.

L. TRANSGENIC ANIMAL APPLICATIONS

In accordance with the non-parenteral administration of the presentinvention, the gene delivery vehicles, particularly those comprised ofunencapsidated nucleic acid, may be complexed with a polycationicmolecule to provide polycation-assisted non-parenteral administration.Such a method of gene delivery facilitates delivery of a gene viamediation by a physical particle comprised of multiple components thataugment the efficiency and specificity of the gene transfer. Inparticular, polycationic molecules, such as polylysine and histone, havebeen shown to neutralize the negative charges on a nucleic acid moleculeand to condense the molecule into a compact form. This form of moleculeis transferred with high efficiency in cells, apparently through theendocytic pathway. The uptake in expression of the nucleic acid moleculein the host cell results after a series of steps, as follows: (1)attachment to cell surface; (2) cell entry via endocytosis or othermechanisms; (3) cytoplasmic compartment entry following endosomerelease; (4) nuclear transport; and (5) expression of the nucleic acidmolecule carried by the gene delivery vehicle. In a further preferredembodiment, multi-layer technologies are applied to thepolycation-nucleic acid molecule complex to facilitate completion of oneor more of these steps. For example, a ligand such asasialoglycoprotein, transferrin, and immunoglobulin may be added to thecomplex to facilitate binding of the cell complex to the cell surface,an endosomal disruption component (e.g., a viral protein, a fusogenicpeptide such as the n-terminus of the influenza virus hemaglutinin or aninactivated virus) is added to facilitate the release of DNA from theendosome, or a nuclear protein (or a peptide containing a nuclearlocalization signal) is added to facilitate the transport of the DNAinto the nucleus. In a further preferred embodiment, the compositioncomprising the complex includes inactivated adenovirus particles(Curiel, D. T., et al., PNAS 88: 8850-8854, 1991; Cristiano, R. J., PNAS90: 2122-2126 1993; Cotten, M., et al., PNAS 89: 6094-6098 1992; Lozier,J. N., et al., Human Gene Therapy 5: 313-322, 1994; Curiel, D. T., etal., Human Gene Therapy 3: 147-154, 1992; Plank, C. et al., BioconjugateChem. 3: 533-539, 1992; Wagner E. et al., PNAS 88: 4255-4259, 1992). Theassorted components comprising the multi-layer complex may be varied asdesired, so that the specificity of the complex for a given tissue, orthe gene expressed from the gene delivery vehicle, may be varied tobetter suit a particular disease or condition.

As noted above, various methods may be utilized to administer genedelivery vehicles of the present invention, including nucleic acidswhich encode the immunogenic portion(s) discussed above, to warm-bloodedanimals such as humans, directly. Suitable methods include, for example,various physical methods such as direct DNA injection (Acsadi et al.,Nature 352:815-818, 1991), and microprojectile bombardment (Williams etal., PNAS 88:2726-2730, 1991).

Within an in vivo context, the gene delivery vehicle can be injectedinto the interstitial space of tissues including muscle, brain, liver,skin spleen or blood (see, WO 90/11092). Administration may also beaccomplished by intravenous injection or direct catheter infusion intothe cavities of the body (see, WO 93/00051), discussed in more detailbelow.

It is generally preferred that administration of the gene deliveryvehicles at multiple sites be via at least two injections. In thisregard, suitable modes of administration include intramuscular,intradermal and subcutaneous injections with at least one of theinjections preferably being intramuscular. In particularly preferredembodiments, two or more of the injections are intramuscular. However,although administration via injections is preferred, it will be evidentthat the gene delivery vehicles may be administered through multipletopical or separate ocular administrations. Further, a number ofadditional routes are suitable for use within the present invention whencombined with one or more of the routes briefly noted above, includingintraperitoneal, intracranial, oral, rectal, nasal, vaginal andsublingual administration. Methods of formulating and administering thegene delivery vehicles at multiple sites through such routes would beevident to those skilled in the art and are described in U.S. Ser. No.08/366,788, filed Dec. 30, 1994 under Attorney Docket No. 930049.429 andU.S. Ser. No. 08/367,071, filed Dec. 30, 1994 under Attorney Docket No.930049.441, incorporated herein by reference in their entirety.

M. VETERINARY APPLICATIONS

From the description provided herein, those skilled in the art willappreciate that the alphavirus vector constructs, recombinant alphavirusparticles, and eukaryotic layered vector initiation systems provided bythe present invention can also be readily utilized in non-human animal(e.g., veterinary) applications. Such applications may includeprophylactics (e.g., vaccines), immunotherapeutics, and palliatives.Within such aspects, compositions and methods are provided foradministering an alphavirus vector construct, recombinant alphavirusparticle, or eukaryotic layered vector initiation system which iscapable of preventing, inhibiting, stabilizing or reversing infectiousdiseases in non-human animals.

Specifically, within one aspect of the prevent invention, compositionsand methods are provided for stimulating an immune response (eitherhumoral or cell-mediated) to a pathogenic agent, such that thepathogenic agent is either killed or inhibited. Representative examplesof pathogenic agents of veterinary importance include bacteria, fungi,parasites and viruses.

More specifically, sequences which encode immunoreactive polypeptides ofthe pathogenic agents may, in certain embodiments, be chosen from agroup that includes the Bunyaviridae (e.g., Rift Valley Fever virus(Giorgi et al., Virology 180:738-753, 1991; Collett et al., Virology144:228-245, 1985)), Paramyxoviridae (e.g., Newcastle disease virus(Millar et al., J. Gen. Virol. 69:613-620, 1988; Chambers et al., Nucl.Acid. Res. 14:9051-9061, 1986; Schaper et al., Virology 165:291-295,1988), and canine distemper virus (Curran et al., J. Gen. Virol.72:443-447, 1991; Barrett et al., Virus Res. 8:373-386, 1987; Bellini etal., J. Virol. 58:408-416, 1986)), Togaviridae (e.g., WEE virus (Weaveret al., Virology 197:375-390, 1993), EEE virus (Chang et al., J. Gen.Virol. 68:2129-2142, 1987), and VEE virus (Kinney et al., Virology152:400-413, 1986)), Rhabdoviridae (e.g., vesicular stomatitus virus(Gill et al., Virology 150:308-312, 1986; Gallione et al., J. Virol.46:162-169, 1983; Banerjee et al., Virology 137:432-438, 1984), andrabies virus (Tordo et al., Nucl. Acid. Res. 14:2671-2683, 1986;Hiramatsu et al., Virus Genes 7:83-88, 1993; Kieny et al., Nature312:163-166, 1984)), Coronaviridae (e.g., transmissable gastroenteritisvirus (Britton et al., Molec. Micro. 2:89-99, 1988; Godet et al.,Virology 188:166-175, 1992; Jackwood et al., Adv. Exp. Med. and Biol.342:43-48, 1993), and feline infectious peritonitis virus (Reed et al.,Adv. Exp. Med. and Biol. 342:17-21, 1993)), Reoviridae (e.g., porcinerotavirus (Burke et al., J. Gen. Virol. 75:2205-2212, 1994; Nishikawa etal., Nucl. Acid. Res. 16:11847, 1988)), Orthomyxoviridae (e.g. equineinfluenza (Gibson et al., Virus Res. 22:93-106, 1992; Dale et al.,Virology 155:460-468, 1986)), Picornaviridae (e.g., FMD virus (Graham etal., Virology 176:524-530, 1990; Brown et al., Gene 75:225-233, 1989;Fross et al., Nucl. Acid. Res. 12:6587-6601, 1984)), and Herpesviridae(e.g., equine herpesvirus (Crabb et al., J. Gen. Virol. 72:2075-2082)).

In other embodiments, the sequences which encode immunoreactivepolypeptides of the pathogenic agents may be chosen from a group thatincludes the agents of coccidiosis (e.g., Eimeria Acervulina, E.tenella, E. maxima (Talebi et al., Infect. Immun. 62:4202-4207, 1994;Pasamotites et al., Mol. Biochem. Parasit. 57:171-174, 1993; Tomely etal., Mol. Biochem. Parasit. 49:277-288, 1991; Castle et al., J. ofParasit. 77:384-390, 1991; Jenkins et al., Exp. Parasit. 70:353-362,1990)), anaplasmosis (e.g., Anaplasma marginale (McGuire et al., Vaccine12:465-471, 1994; Palmer et al., Infect. Immun. 62:3808-3816, 1994;Oberle et al., Gene 136:291-294, 1993; Barbet et al., Infect. Immun.59:971-976, 1991; Barbet et al., Infect. Immun. 55:2428-2435; 1987)),babesiosis (e.g., Babesia bovis (Suarez et al., Infect. Immun.61:3511-3517, 1993; Hines et al., Mol. Biochem. Parasit. 55:85-94, 1992;Jamer et al., Mol. Biochem. Parasit. 55:75-83, 1992; Suarez et al., Mol.Biochem. Parasit. 45:45-52, 1991)), theileriosis (e.g. Theileria parva(Nene et al., Mol. Biochem. Parasit. 51:17-27, 1992; Iams et al., Mol.Biochem. Parasit. 39:47-60, 1990)), malaria (e.g., Plasmodium falciparum(Haeseleer et al., Mol. Biochem. Parasit. 57:117-126, 1993)),salmonellosis (Salmonella typhimurium and S. dublin), bovine and ovinemastitis (Staphylococcus aureus), bovine tuberculosis (Mycobacteriumbovis), pseudotuberculosis (Yersinia pseudotuberculosis),coccidioidomycosis (Coccidioides immitis), cryptococcosis (Cryptococcusneoformans), anthrax (Bacillus anthracis), brucellosis (Brucella abortusand B.suis), and leptospirosis (Leptospira interrogans and L.biflexa).

To illustrate this aspect in more detail, methods used on constructingrecombinant alphavirus vectors and eukaryotic layered vector initiationsystems containing these sequences for veterinary application aredescribed for two of the above pathogenic agents (one viral and oneparasitic). The construction of additional alphavirus vectors andeukaryotic layered vector initiation systems is readily accomplished bythose skilled in the art, based on the following methodologies and usingsequences from other related or non-related pathogenic agents. In thecase of foot-and-mouth disease virus (FMDV), a cassette comprising eachof the four P1 capsid proteins (1A, 1B, 1C, 1D) and the 3C proteaseresponsible for their post-translational cleavage is obtained asplasmids MR1 and MR2 from Graham et al. (Virology 176:524-530, 1990).Plasmid MR1 or MR2 is digested with the enzymes HindIII and DraI toremove the FMDV P1 cassette, followed by the fill-in of the HindIIterminus with Klenow, and purification from a 1% agarose gel usingGENECLEAN™. Plasmid vectors pKSSINBV and pVGELVIS-SINBV (see Example 3)are digested with XhoI and the terminal also made blunt using Klenow,followed by treatment with CIAP and purification from a 1% agarose gelusing GENECLEAN™. The purified fragments are subsequently ligated togenerate the alphavirus vector construct pKSSIN-FMDV and eukaryoticlayered vector initiation system plasmid pVGELVIS-FMDV. The purifiedFMDV sequences are also readily inserted into any of the other vectorconstructs described in this invention (see Example 3). Packaging of theFMDV-containing alphavirus vector construct pKSSIN-FMDV can beaccomplished as described in Example 7.

For construction of a recombinant alphavirus vector construct oreukaryotic layered vector initiation system comprising sequences from apathogenic agent of anaplasmosis, the major surface protein 2 (MSP-2).of A. marginale is obtained by PCR amplification from plasmid pCKR11.2(Palmer et al., Infect. Immun. 62:3808-3816, 1994) using the followingoligonucleotide pair, each containing a flanking XhoI site:

forward primer (AM-MSP-2F):

5′-TATATCTCGAGACCACCATGAGTGCTGTAAGTAATAGGAAGC   (SEQ. ID NO. 115)

reverse primer (AM-MSP-2R):

5′-TATATCTCGAGCTAGAAGGCAAACCTAACACCCAAC   (SEQ. ID NO. 116)

A standard three temperature cycling protocol is performed as describedpreviously using THERMALASE™ thermostable polymerase, theoligonucleotide pair, and plasmid pCKR11.2 as template. Followingamplification, the MSP-2 amplicon is purified using GENECLEAN™, digestedwith XhoI, and re-purified with GENECLEAN™. Plasmid vectors pKSSINBV andpVGELVIS-SINBV (see Example 3) also are digested with XhoI, followed bytreatment with CIAP and subsequent ligation to the MSP-2 fragment togenerate the alphavirus vector construct pKSSIN-MSP2 eukaryotic layeredvector initiation system plasmid pVGELVIS-MSP2. The purified MSP-2sequences are also readily inserted into any of the other vectorconstructs described elsewhere in this specification (e.g., Example 3).Packaging of the MSP-2-containing alphavirus vector constructpKSSIN-MSP2 can be accomplished as described in Example 7.

Example 4

A. INSERTION OF ADENOVIRUS EARLY REGION E3 GENE INTO SINDBIS VECTORS

In order to inhibit the host CTL response directed against viralspecific proteins expressed in vector infected cells, in applicationswhere repeated administration of the therapeutic is desired, theAdenovirus type 2 (Ad 2) E3/19K gene ATCC No. VR-846 is cloned into thepKSSINdlJRsjrc plasmid, immediately downstream from the junction regioncore. Briefly, Ad 2 is propagated in a permissive cell line, for exampleHeLa or Vero cells, and after evidence of cytopathologic effects,virions are purified from the cell lysate, and the Ad 2 DNA is purifiedfrom the virus.

The Ad 2DNA E3/19K gene, including the amino terminal signal sequence,followed by the intraluminal domain and carboxy terminal cytoplasmictail which allows the E3 19K protein to embed itself in the endoplasmicreticulum, is located between viral nucleotides 28,812 and 29,288.Isolation of the Ad 2 E3 19K gene from the viral genomic DNA isaccomplished by PCR amplification, with the primer pair shown below:

Ad 2 E3 Forward primer (Ad 2 nucleotides 28,812-28,835):

5′-TAT ATC TCC AGA TGA GGT ACA TGA TTT TAG GCT TG-3′  (SEQ. ID NO. 56)

Ad 2 E3 Reverse primer (Ad 2 nucleotides 29,241-29,213):

5′-TAT ATA TCG ATT CAA GGC ATT TTC TTT TCA TCA ATA AAA C   (SEQ. ID NO.57)

In addition to the Ad 2 complementary sequences, both primers contain afive nucleotide “buffer sequence” at their 5′ ends for efficient enzymedigestion of the PCR amplicon products. This sequence in the forwardprimer is followed by the Xho I recognition site, and in the reverseprimer this sequence is followed by the Cla I recognition site. Thus, inthe 5′ to 3′ direction, the E3/19K gene is flanked by Xho I and Cla Irecognition sites. Amplification of the E3/19K gene from Ad 2 DNA isaccomplished with the following PCR cycle protocol:

Temperature (° C.) Time (Min.) No. Cycles 94 2 1 94 0.5 55 0.17 5 72 3.594 0.5 30 70 3.5 72 10 10

Following amplification, the 451 bp amplicon is purified on a 1.5%agarose gel, and digested with the Xho I and Cla I enzymes.pKSSINdlJRsjrc plasmid is partially digested with ClaI. Plasmid that hasbeen digested only once is isolated by gel electrophoresis then digestedwith XhoI. The large fragment is isolated by gel electrophoresis andligated to the digested PCR amplicon. This clone is designatedpKSSINdlJRsjrcAdE3. Using the same cloning strategy, the Ad 2 E3/19Kgene may be inserted into any of the modified synthetic junction regionvectors or ELVIS vectors described in Example 3.

B. INSERTION OF THE HUMAN CYTOMEGALOVIRUS H301 GENE INTO SINDBIS VECTORS

In order to inhibit the host CTL directed response against viralspecific proteins expressed in vector infected cells in applicationswhere repeated administration of the therapeutic is desired, the humancytomegalovirus (HCMV) H301 gene is cloned into the pKSSINdlJRsjrcplasmid, immediately downstream from the junction region core.

Briefly, HCMV strain AD169 (ATCC No. VR-538), is propagated in apermissive cell line, for example primary human foreskin fibroblasts(HFF) (GIBCO/BRL, Gaithersburg, Md.), and after evidence ofcytopathologic effects, virions are purified from the cell lysate.Subsequently, HCMV DNA is purified from the virons.

The HCMV H301 gene is located between viral nucleotides 23,637 and24,742. Isolation of the HCMV H301 gene from the viral genomic DNA isaccomplished by PCR amplification, with the primer pair shown below:

HCMV H301 Forward primer (buffer sequence/Xho I site/HCMV nucleotides23,637-23,660):

5′-TAT ATC TCC AGA TGA TGA CAA TGT GGT GTC TGA CG-3′  (SEQ. ID NO. 58)

HCMV H301 Reverse primer (buffer sequence/Cla I site/HCMV nucleotides24,744-24,722):

5′-TAT ATA TCG ATT CAT GAC GAC CGG ACC TTG CG-3′  (SEQ. ID NO. 59)

In addition to the HCMV H301 gene complementary sequences, both primerscontain a five nucleotide “buffer sequence” at their 5′ ends forefficient enzyme digestion of the PCR amplicon products. This sequencein the forward primer is followed by the Xho I recognition site, and inthe reverse primer this sequence is followed by the Cla I recognitionsite. Thus, in the 5′ to 3′ direction, the HCMV H301 gene is flanked byXho I and Cla I recognition sites. Amplification of the HCMV H301 genefrom HCMV DNA is accomplished with the following PCR cycle protocol:

Temperature (° C.) Time (Min.) No. Cycles 94 2 1 94 0.5 55 0.17 5 72 3.594 0.5 30 70 3.5 72 10 10

Following amplification, the 1,129 bp amplicon product is purified on a1.0% agarose gel, and subsequently digested with the Xho I and Cla Ienzymes and ligated into the CIAP treated pKSSINdlJRsjrc plasmid,previously digested with Xho I and Cla I as described above. This cloneis designated pKSSINdlJRsjrcH301. Using the same cloning strategy, theHCMV H301 gene is inserted into all of the modified synthetic junctionregion vectors and all of the ELVIS vectors described in Example 3.

Example 5 Expression of Multiple Heterologous Genes From Sindbis Vectors

The plasmid pBS-ECAT (Jang et al., J. Virol 63:1651, 1989) includes the5′ nontranslated region of Encephalomycarditis (EMCV) from nts 260-848of the viral genome, which contains the internal ribosome entry site(IRES). EMCV nucleotides 260-827 are amplified from pBS-ECAT by PCR,using the following primer pair:

EMCV IRES Forward primer A (For insertion next to disabled junctionregion in vector pKSSINBVdlJR at Apa I site):

5′-TAT ATG GGC CCC CCC CCC CCC CCC AAC G-3′  (SEQ. ID NO. 60)

EMCV IRES Forward primer B (For insertion between heterologous genesterminating with Cla I sites and initiating with Nco I sites):

5′-TAT ATA TCG ATC CCC CCC CCC CCC CCA ACG-3′  (SEQ. ID NO. 61)

EMVC IRES Reverse Primer (To be used with either primers A or B):

5′-TAT ATC CAT GGC TTA CAA TCG TGG TTT TCA AAG G-3′  (SEQ ID NO. 62)

The amplicon resulting from amplification with the forward primer A andthe reverse primer is flanked by Apa I and Nco I recognition sites,inside a 5 bp ‘buffer sequence’.

The amplicon resulting from amplification with the forward primer B andthe reverse primer is flanked by Cla I and Nco I recognition sites,inside a 5 bp ‘buffer sequence’.

Amplification of the EMCV IRES sequence from the pBS-ECAT plasmid isaccomplished with the following PCR cycle protocol:

Temperature (° C.) Time (Min.) No. Cycles 94 2 1 94 0.5 55 0.17 5 72 3.594 0.5 30 70 3.5 72 10 1

In a similar manner, the ATG corresponding to the start codon of theheterologous gene to be inserted immediately downstream of the EMVC IRESinsert is modified to contain an NcoI site (CCATGG) while the 3′ end ismodified to contain a ClaI site.

For insertion into the pKSSINBVdlJR vector, the 589 bp ECMV-IRESamplicon is digested with ApaI and NcoI, purified on a 1% agarose gel.The heterologous gene amplicon is digested with NcoI and ClaI andpurified in a similar manner. Both fragments are ligated into the CIAPtreated vector digested with Apa I and ClaI as described in example 4.

For insertion into the pKSSINBV or pKSSINBVdlJRsjrc vectors betweenheterologous genes, the 589 bp amplicon is digested with Cla I and NcoI, purified on a 1% agarose gel, and ligated into the bicistronicheterologous gene vector digested with Cla I and Nco I and treated withCIAP. In a bicistronic heterologous gene configuration, the 3′ end ofthe upstream heterologous gene is modified to terminate in a Cla Irecognition site. The ATG corresponding to the start codon of the seconddownstream heterologous gene to be inserted immediately downstream ofthe EMCV IRES insert is modified to contain an Nco I site (CCATGG).Thus, from 5′ to 3′, the order of components is: pKSSINBV orpKSSINBVdlJRsjrc-gene #1-Cla/Nco EMCV IRES gene #2-3′ SIN. Insertioninto all of the modified junction region vectors described in Example 2and and all of the ELVIS vectors described in Example 3 follows thestrategy given here for the pKSSINBV or pKSSINBVdlJRsjrc vectors.

The pKSSINBVdlJR vector containing a bicistronic heterologousconfiguration is constructed with each of the EMCV IRES ampliconsdescribed above. The first EMCV IRES amplicon is flanked by Apa I andNco I sites and is inserted immediately downstream of the disabledjunction region at the Apa I site, as described above. This EMCV IRESsequence is followed by the first heterologous gene, which terminates ina Cla I recognition site. The first heterologous gene is followed by thesecond EMCV IRES sequence, using the amplicon flanked by Cla I and Nco Irecognition sites. The second heterologous gene follows the second EMCVIRES sequence. Thus, from 5′ to 3′, the order of components is:SINBVdlJR-Apa/Nco EMCV IRES gene #1-Cla/Nco EMCV IRES gene #2-3′ SIN.

The plasmid pP2-5′ (Pelletier et al., Mol. Cell Biol. 8:1103, 1988)includes the 5′ nontranslated region of the poliovirus P2/Lansing strainfrom nucleotides 1-1,872 of the viral genome, which contains the polioIRES. Poliovirus nucleotides 320-631 are amplified from pP2-5′ by PCR,using the following primer pair:

Polio IRES Forward primer A (For insertion next to disabled junctionregion in vector pKSSINBVdlJR at Apa I site):

5′-TAT ATG GGC CCT CGA TGA GTC TGG ACG TCC CTC-3′  (SEQ. ID NO. 63)

Polio IRES Forward primer B (For insertion between heterologous genesterminating with Cla I sites and initiating with Nco I sites):

5′-TAT ATA TCG ATT CGA TGA CTC TGG ACG TTC CTC-3′  (SEQ. ID NO. 64)

Polio IRES Reverse Primer (To be used with either primers A or B):

5′-TAT ATC CAT GGA TCC AAT TTG CTT TAT GAT AAC AAT C-3′  (SEQ. ID NO.65)

The amplicon resulting from PCR with the Polio IRES forward primerA/reverse primer pair shown above is flanked by Apa I and Nco Irecognition sites, inside a 5 bp ‘buffer sequence’. The ampliconresulting from PCR with the Polio IRES forward primer B/reverse primerpair is shown above is flanked by Cla I and Nco I recognition sites,inside a 5 bp ‘buffer sequence’. Amplification of the polio IRESsequence from the pP2-5′ plasmid is accomplished with the PCR protocolshown in Example 5. In a similar manner, the ATG corresponding to thestart codon of the heterologous gene to be inserted immediatelydownstream of the Polio IRES insert is modified to contain an NcoI site(CCATGG) while the 3′ end is modified to contain a ClaI site.

For insertion into the pKSSINBVdlJR vector, the 333 bp Polio-IRESamplicon is digested with Apa I and Nco I and purified on a 1.5% agarosegel. The heterologous gene amplicon is digested with NcoI and ClaI andpurified in a similar manner. Both fragments are ligated into the CIAPtreated vector digested with Apa I and ClaI as described in example 4.

For insertion into the pKSSINBV or pKSSINBVdlJRsjrc vectors betweenheterologous genes, the 333 bp amplicon is digested with Cla I and NcoI, purified on a 1.5% agarose gel, and ligated into the bicistronicheterologous gene vector digested with Cla I and Nco I and treated withCIAP. In a biscistronic heterologous gene configuration, the 3′ end ofthe upstream heterologous gene is modified to terminate in a Cla Irecognition site. The ATG corresponding to the start codon of the seconddownstream heterologous gene to be inserted immediately downstream ofthe polio IRES insert is modified to contain an Nco I site (CCATGG).Thus, from 5′ to 3′, the order of components is: pKSSINBV orpKSSINBVdlJRsjrc-gene #1-Cla/Nco polio IRES gene #2-3′ SIN. Insertioninto all of the modified junction region vectors and all of the ELVISvectors described in Example 3 follows the strategy given here for thepKSSINBV or pKSSINBVdlJRsjrc vectors.

The pKSSINBVdlJR vector containing a bicistronic heterologousconfiguration is constructed with each of the polio IRES ampliconsdescribed above. The first polio IRES amplicon is flanked by Apa I andNco I sites and is inserted immediately downstream of the disabledjunction region at the Apa I site, as described above. This polio IRESsequence is followed by the first heterologous gene, which terminates ina Cla I recognition site. The first heterologous gene is followed by thesecond polio IRES sequence, using the amplicon flanked by Cla I and NcoI recognition sites. The second heterologous gene follows the secondpolio IRES sequence. Thus, from 5′ to 3′, the order of components is:SINBVdlJR-Apa/Nco polio IRES gene #1-Cla/Nco EMCV IRES gene #2-3′ SIN.

The 220 bp BiP cDNA, corresponding to the 5′ leader region of the humanimmunoglobulin heavy-chain binding protein mRNA, is amplified from aplasmid containing the 5′ noncoding region of the BiP gene, pGEM5ZBiP5′(provided by P. Sarnow, University of Colorado Health Sciences Center),using PCR. The sequence corresponding to BiP cDNA was determinedoriginally in the bacteriophage lambda hu28-1 clone of the human GRP78gene (Ting and Lee, DNA 7:275-286, 1988). The forward primer to be usedin the PCR reaction varies, depending on the Sindbis vector into whichthe BiP cDNA is inserted. The reverse primer for the PCR reaction is thesame for all Sindbis vectors. Amplification of the BiP cDNA sequencefrom pGEM5ZBiP5′ from the plasmid for insertion into the Sindbis vectorpKSSINBVdlJR, immediately downstream of the disabled junction region, isaccomplished by amplification with the following forward primer:

5′-TAT ATG GGC CCG GTC GAC GCC GGC CAA GAC-3′  (SEQ. ID No. 66)

In addition to the BiP cDNA complementary sequences, beginning atnucleotide 12, the primer contains a five nucleotide ‘buffer sequence’at its 5′ end for efficient enzyme digestion of the PCR ampliconproducts. This sequence is followed by the Apa I recognition site.

Amplification of the BiP cDNA sequence from the pGEM5ZBiP5′ plasmid forinsertion into the Sindbis vectors pKSSINBV, or pKSSINBVdlJRsjrc, isaccomplished by amplification with the following forward primer shownbelow. For these vectors, the BiP cDNA is inserted between twoheterologous genes, which are placed in the region corresponding to theSindbis structural genes.

5′-TAT ATA TCG ATG GTC GAC GCC GGC CAA GAC-3′  (SEQ. ID NO. 67)

In addition to the BiP cDNA complementary sequences, beginning atnucleotide 12, the primer contains a five nucleotide ‘buffer sequence’at its 5′ end for efficient enzyme digestion of the PCR ampliconproducts. This sequence is followed by the Cla I recognition site.

The reverse primer for amplification of the BiP cDNA sequence from thepGEM5ZBiP5′ plasmid for insertion into the Sindbis vectors pKSSINBVdlJR,pKSSINBV, or pKSSINBVdlJRsjrc, is:

5′-TAT ATC CAT GGT GCC AGC CAG TTG GGC AGC AG-3′  (SEQ. ID NO. 68)

In addition to the BiP cDNA complementary sequences, beginning atnucleotide 12, the reverse primer contains a five nucleotide ‘buffersequence’ at its 5′ end for efficient enzyme digestion of the PCRamplicon products. This sequence is followed by the Nco I recognitionsite. Amplification of the BiP cDNA from the pGEM5ZBiP5′ is accomplishedwith PCR protocol that are described above. In a similar manner, the ATGcorresponding to the start codon of the heterologous gene to be insertedimmediately downstream of the BiP IRES insert is modified to contain anNcoI site (CCATGG) while the 3′ end of modified to contain a ClaI site.

For insertion into the pKSSINBVdlJR vector, the 242 bp BiP IRES ampliconis digested with Apa I and Nco I and purified on a 2% agarose gel. Theheterologous gene amplicon is digested with NcoI and ClaI and purifiedin a similar manner. Both fragments are ligated into the CIAP treatedvector digested with Apa I and ClaI as described in example 4.

For insertion into the pKSSINBV or pKSSINBVdlJRsjrc vectors betweenheterologous genes, the 242 bp BiP IRES amplicon is digested with Cla Iand Nco I, purified on a 2% agarose gel, and ligated into thebicistronic heterologous gene vector digested with Cla I and Nco I andtreated with CIAP. In a biscistronic heterologous gene configuration,the 3′ end of the upstream heterologous gene is modified to terminate ina Cla I recognition site. The ATG corresponding to the start codon ofthe second downstream heterologous gene to be inserted immediatelydownstream of the BiP cDNA insert is modified to contain an Nco I site(CCATGG). Thus, from 5′ to 3′, the order of components is: pKSSINBV orpKSSINBVdlJRsjrc-gene #1-Cla/Nco BiP-gene #2-3′ SIN. Insertion into allof the modified junction region vectors described in Example 2, and intoall of the ELVIS vectors described in example 3, follows the strategygiven here for the pKSSINBV or pKSSINBVdlJRsjrc vectors.

The pKSSINBVdlJR vector containing a bicistronic heterologousconfiguration is constructed with each of the BiP cDNA ampliconsdescribed above. The first BiP cDNA amplicon is flanked by Apa I and NcoI sites and is inserted immediately downstream of the disabled junctionregion at the Apa I site, as described above. This BiP sequence isfollowed by the first heterologous gene, which terminates in a Cla Irecognition site. The first heterologous gene is followed by the secondBiP cDNA sequence, using the amplicon flanked by Cla I and Nco Irecognition sites. The second heterologous gene follows the second BiPsequence. Thus, from 5′ to 3′, the order of components is:SINBVdlJR-Apa/Nco BiP-gene #1-Cla/Nco BiP-gene #2-3′ SIN.

Sequences which promote ribosomal readthrough are placed immediatelydownstream of the disabled junction region in the pKSSINBVdlJR vector,which allows ribosomal scanning in mRNA from non-structural genetermination to the heterologous genes. The heterologous proteins areexpressed from genomic length mRNA by ribosomal scanning. This extendsthe life of the infected target cell because no subgenomic transcriptionoccurs in cells infected with this vector. Further, these same ribosomalscanning sequences are placed between heterologous genes contained inpolycistronic subgenomic mRNAs. The ribosomal spanning sequence to beused in the pKSSINBVdlJR vector and between heterologous genes in thepolycistronic mRNA region is:

5′-TTA ATT AAC GGC CGC CAC CAT GG-3′  (SEQ. ID NO. 69)

The boldfaced codons refer to the ochre stop codon and AUG start codon,respectively. The bases underlined surrounding the stop codon refer tothe Pac I recognition site and the bases underlined surrounding thestart codon refer to the Nco I recognition site. The intercistronicdistance of 15 bp between the start and stop codons allows efficientribosomal readthrough, as shown previously (Levine et al., Gene108:167-174, 1991). The sequences surrounding the ATG start codon frombases −9 to +1 conform to the Kozak consensus sequence for efficienttranslational initiation (Kozak, Cell 44:283-292, 1986). Where possible,the 3′ terminal nucleotide corresponding to the carboxy terminal aminoacid is changed to T, by site-directed mutagenesis. Also, the 5′terminal nucleotide corresponding to the amino terminal amino acid inthe downstream cistron is changed to G, by site-directed mutagenesis.

Insertion of the intercistronic sequence between heterologous genes, ordownstream of the disabled junction region in vector pKSSINBVdlJR,modified as described above, is accomplished by insertion of thedouble-stranded oligonucleotide pair shown below, into compatible PacI/Nco I ends:

Read through sense Oligonucleotide:

5′-TAA CGG CCG CCA C-3′  (SEQ. ID NO. 70)

Read through antisense Oligonucleotide:

5′-CCA TGG TGG CGG CCG TTA AT-3′  (SEQ. ID NO. 71)

The oligonucleotides above are mixed in equal molar quantities in thepresence of 10 mM MgCl₂, heated at 95° C. for 5 min, then allowed tocool slowly to room temperature, yielding the desired intercistronicsequence flanked by Pac I and Nco I sites. The intercistronic sequenceis then ligated into the appropriate vector containing Pac I and Nco Icompatible sites.

Another aspect of the present invention to enable expression of multipleheterologous genes in eukaryotic layered vector initiation systems isbased on the use of alternate splicing signals. In this configuration, asplice door sequence is inserted immediately downstream of the junctionregion promoter, followed by one or more heterologous genes, each ofwhich is preceded by a splice acceptor sequence. As such, multiplesplice acceptor/heterologous gene inserts may be arrayed 3′ to oneanother. This creates a system whereby multiple heterologous genes areexpressed from a single eukaryotic layered vector initiation systemtranscript, which is processed alternately at each splice acceptor siteto give rise to individual autocatalytic RNAs encoding an individualheterologous gene. In such a system, levels of expression for eachheterologous gene is controlled independently by altering the nucleotidesequence of the splice acceptor site. In addition, multiple splicedonor/acceptor sites may be engineered into the system. Finally, tissuespecific splice donor/acceptor sequences may be utilized in such asystem to control the expression in specific tissues.

Example 6 Expression of Multiple Heterologous Genes by Copackaging

The ability to copackage multiple RNA molecules in the same alphavirusvector particle can be useful for the expression of multipleheterologous gene products from a single alphavirus vector particle. Inaddition, this concept can also be adapted in order to allow very largegenes to be carried on RNA molecules separate from the alphavirus vectorRNA containing the nonstructural genes, thus avoiding the need topackage very long vector RNA molecules.

In order to accomplish such copackaging, all RNA fragments must containa 5′ sequence which is capable of initiating transcription of analphavirus RNA, an alphavirus RNA polymerase recognition sequence forminus-strand synthesis, and at least one copy of the RNA packagingsequence. At least one of the RNA fragments also must contain sequenceswhich code for the alphavirus non-structural proteins. Within preferredembodiments of the invention, one or more of the RNA fragments to becopackaged also will contain a viral junction region followed by aheterologous gene.

A. Construction of Copackaged Expression Cassettes for Expression ofMultiple Heterologous Genes

In order to demonstrate the feasibility of copackaging to allow for theexpression of multiple heterologous genes, two vector constructs arecreated. The first construct consists of a 5′ sequence that is capableof initiating transcription of Sindbis virus RNA, Sindbis RNA sequencesrequired for packaging, sequences encoding the synthesis ofnonstructural proteins 1-4, a Sindbis junction region, the luciferasegene, and Sindbis 3′ sequences required for synthesis of the minusstrand RNA. The second construct consists of a 5′ sequence that iscapable of initiating transcription of a Sindbis virus, Sindbissequences required for packaging, a Sindbis Junction region, Sequencesencoding the LacZ gene, and Sindbis 3′ sequences required for synthesisof the minus strand RNA. RNA transcripts of these constructs transfectedinto a packaging cell line are copackaged to produce a vector particlecapable of transferring expression of both luciferase andβ-galactosidase into the same eukaryotic cell.

The β-galactosidase reporter gene is inserted into the Sindbis BasicVector (pKSSINBV) followed by deletion of a portion of the Sindbisnon-structural proteins from the vector. RNA from this construct iscotransfected with RNA from Sindbis Luciferase Vector (pKSSINBV-luc) andis copackaged by one of the methods described in Example 7. Infection offresh BHK-21 cells with vector particles containing the copackaged RNAexpression cassettes should result in the expression of both luciferaseand β-galactosidase in the same cell.

B. Construction of a β-Galactosidase Expression Cassette

pKSSINBV-Linker is digested with the enzyme Cas I, which cleavesimmediately after the Sindbis 3′-end and poly A sequence. The digestedfragment is treated with alkaline phosphatase and purified usingGeneclean. Two 12 mer oligonucleotides,

5′ GGTTTAAACAGGAGCT 3′  (SEQ. ID NO. 72)

5′ CCTGTTTAAACCAGCT 3′  (SEQ. ID No. 73)

which form the Pme I site with SacI compatible ends when hybridized,were phosphorylated and ligated into the SacI digested vector. Thisconstruct is known as pKSSINBV-Linker-PmeI. The Pme I recognition siteis substituted for the Sac I site in order to create a site forlinearization of the plasmid prior to SP6 transcription. The lacZ genecontains several Sac I sites. pKSSINBV-Linker-PmeI is digested with PmlI and Bcl I followed by purification with GENECLEAN. The lacZ gene isobtained by digestion of pSV β-galactosidase vector DNA (Promega Corp.,Madison, Wis.) with the enzyme HindIII. The digest is blunt-ended withKlenow DNA polymerase and dNTPs. The Klenow is heat killed and theplasmid is further digested with Bam HI and Xmn I. Xmn I reduces thesize of the remaining vector fragment to simplify gel purification ofthe lacZ fragment. The 3.7 kbp lacZ fragment is purified from a 1%agarose gel and ligated into the Pml I/Bcl I digestedpKSSINBV-Linker-PmeI fragment. This construct is known as pKSSINBV-lacZ.pKSSINBV-lacZ is digested with Bsp EI and religated under diluteconditions. This results in the removal of the Sindbis nonstructuralproteins between nt#422-7054. This Sindbis construct is known aspKSSINBVdlNSP-lacZ.

pKSSINBVdlNSP-lacZ and pKSSINBV-luc are linearized with Pme I and Sac I,respectively, and SP6 transcripts are prepared as described in Example3. These RNA transcripts are cotransfected into packaging cells thatexpress the Sindbis structural proteins by one of the mechanismsdescribed in Example 7. Each RNA transcript contains a 5′ sequence thatis capable of initiating transcription of a Sindbis virus, RNA sequencesrequired for packaging, a Sindbis junction region, a reporter gene, andSindbis 3′ sequences required for synthesis of the minus strand RNA. ThepKSSINBV-luc transcript also contains the Sindbis non-structuralproteins. In cotransfected cells, both RNA transcripts are replicatedand some viral particles will contain both RNA transcripts copackagedinto the same particle. Infection of fresh cells with the copackaged RNAparticles will result in cell that express both luciferase andβ-galactosidase.

C. Copackaging of Multiple Expression Cassettes to Increase PackagingCapacity

Large genes such as Factor VIII can benefit from copackaging. Briefly,insertion of the cDNA coding for Factor VIII into the Sindbis BasicVector (pKSSINBV) results in an RNA transcript approaching 16 kb inlength. Because of the increased length, this RNA cannot be replicatedor packaged efficiently. Using approaches described above, the Sindbisnonstructural proteins and the Factor VIII gene could be divided ontoseparate RNA molecules of approximately 8 kb and 9 kb in length, andcopackaged into the same particles.

D. Construction of a Factor VIII Expression Cassette

The pKSSINBV-Linker-PmeI construct is digested with the enzyme Bsp EIand religated under dilute conditions. This results in the removal ofthe Sindbis nonstructural proteins between nt#422-7054. This constructis known as pKSSINBVdlNSP-Linker-PmeI. The pKSSINBVdlNSP-Linker-PmeIconstruct is digested with the enzymes Pml I and Stu I and purified byusing Geneclean. The source of Factor VIII cDNA is clone pSP64-VIII, anATCC clone under the accession number 39812 having a cDNA encoding thefull-length human protein. pSP64-VIII is digested with Sal I, the endsare blunted with T4 DNA polymerase and 50 uM of each dNTP, and the ca.7700 bp. fragment is electrophoresed on a 0.7% agarose/TBE gel andpurified with Geneclean. The 7.7 kb fragment encoding Factor VIII ispurified is purified in a 0.7% agarose gel and subsequently ligated tothe Pml I/Stu I digested pKSSINBVdlNSP-Linker-PmeI fragment. Thisconstruct is known as pKSSINBVdlNSP-Factor VIII.

pKSSINBVdlNSP-Factor VIII and pKSSINBV constructs are linearized withPme I and Sac I, respectively. SP6 transcripts are prepared as describedin Example 3. These RNA transcripts are cotransfected into packagingcells that express the Sindbis structural proteins by one of themechanisms described in Example 7. Both RNA transcripts contain a 5′sequence that is capable of initiating transcription of Sindbis RNA,sequences required for RNA packaging, a Sindbis Junction region, and theSindbis 3′ sequences required for synthesis of the minus strand RNA. Inaddition, the pKSSINBV transcript contains the Sindbis nonstructuralprotein genes, and the pKSSINBVdlNSP-Factor VIII construct contains theFactor VIII gene, but not the Sindbis nonstructural protein genes. Incotransfected cells, both RNA transcripts are replicated and some viralparticles will contain both RNA transcripts copackaged into the samevector particle. Infection of fresh BHK-21 cells with the copackaged RNAwill result in Factor VIII expression only if both RNA molecules arepresent in the same cell.

E. Construction of an Aura Virus Copackaging Vector

To develop Aura virus expression systems analogous to those describedfor Sindbis, stranded techniques known in the art (e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, 1989), as well as specific approaches describedherein, will be utilized for construction. Virus, obtained from theATCC, is propagated on cultured cells, its virion RNA extracted, andcDNA spanning the entire genome synthesized and cloned usingconventional techniques. This cDNA is then used to construct genetransfer vector systems similar in principal to those described above,including, but not limited to, a replicon capable of carrying theheterologous gene(s), packaging cell lines that express the structuralprotein genes, and unique to this system, a separate packaging-competentsubgenomic vector capable of carrying the additional heterologousgene(s). Since Aura virus subgenomic RNA contains a packaging signal,preliminary experiments are performed to identify this sequence, inorder to prevent its inactivation during replacements with heterologousthe gene(s). After identification of the packaging sequence, theindividual elements of this Aura-based system are generated.

A basic replicon vector is constructed to contain the following minimumelements: Aura 5′ sequences necessary for replication, nonstructuralprotein coding regions, a modified or unmodified junction region forsubgenomic mRNA synthesis, a restriction enzyme site for insertion ofheterologous gene(s), one or more copies of the packaging signal, and 3′Aura sequences necessary for replication, including a polyadenylatesequence. An upstream bacteriophage RNA polymerase promoter will beutilized for in vitro transcription of replicon RNA; alternatively, aeukaryotic RNA polymerase promoter will be utilized for transcriptiondirectly from cDNA.

A packaging-competent subgenomic vector is also constructed to containthe following minimum elements: a modified or unmodified junctionregion, a restriction enzyme site for insertion of heterologous gene(s),one or more copies of the packaging signal, and 3′ Aura sequencesnecessary for replication/minus-strand synthesis, including apolyadenylate sequence. The subgenomic vector may, in some cases, beconstructed with the Aura 5′ replication sequences positioned upstreamof the junction region, such that the vector will function as anamplicon. Transcription of subgenomic vector RNA can be accomplished invitro using a bacteriophage RNA polymerase promoter, or cDNA in vivousing a eukaryotic RNA polymerase promoter. Further, the initialtranscript may be of the sense-configuration or of theantisense-configuration.

Packaging cell lines are also constructed as described previously forSindbis vectors, such that mRNA for one or more of the structuralproteins will be transcribed from the junction region and be inducibleby the Aura replicon. In other cases, one or more of the structuralproteins can be expressed under the control of an inducible orconstitutive eukaryotic promoter. In each case, specific inactivatingmutations are made in any packaging sequences present in the structuralprotein genes, in order to prevent encapsidation of these sequences withthe replicon. These mutations should be silent changes, usually at thethird position of the codon, which do not affect the amino acid encoded.

The ability to package multiple heterologous genes can be exploited formany therapeutic applications, which include, but are not limited to,expression of multiple cytokines, multiple CTL epitopes, combinations ofcytokines and CTL epitopes to enhance immune presentation, multiplesubunits of a therapeutic protein, combinations of therapeutic proteinsand antisense RNAs, etc. In addition to its utility for the expressionof multiple heterologous genes, the packaging of subgenomic mRNAs intovirions also enables this vector system for the transfer of extremelylong heterologous sequences. Furthermore, this multipartite approach isuseful in the development of producer cell lines, wherein replicaseproteins and structural proteins are being stably expressed, and anyheterologous gene contained within a subgenomic vector could then bereadily introduced as a stable integrant.

Example 7 Construction of Alphavirus Packaging Cell Lines

A. Selection of Parent Cell Lines for Alphavirus Packaging Cell LineDevelopment

1. Persistently or Chronically Infectable Cells

An important criteria in selecting potential parent cell lines for thecreation of alphavirus packaging cell lines, is the choice of cell linesthat exhibit little or no cytopathological effects, prior to theappropriate production of alphavirus vector particles. This criteria isessential for the development of an alphavirus vector producer cell linewhich can be propagated for long periods of time and used as a stablesource of vector. It is known that alphavirus infection of mostmammalian cells results in cytopathology and lysis of the cell. However,the derivation of packaging cells from various insect cell lines maycircumvent this problem. For example, insect cell lines, such as Aedesalbopictus, Aedes aegypti, Spodoptera frugiperda, and Drosophilamelanogaster cells, may be utilized to construct packaging cell lines.For example, within one embodiment, alphavirus packaging cell lines areprovided using an configuration uses an insect parent cell line, such asthe Aedes albopictus, containing a stably transfected expressioncassette vector which allows for expression of alphavirus structuralproteins under the control of inducible or non-inducible promotersactive in these cell types, and co-expressing a selectable marker.

Recently, a Sindbis virus-induced protein of cellular origin, which hasbeen associated with the down-regulation of Sindbis virus production insome infected Aedes albopictus cells, has been identified and purified(Purification and Characterization of A Sindbis Virus-Induced PeptideWhich Stimulates Its Own Production and Blocks Virus RNA Synthesis, Luo,et al., Virology 194, 44-49, 1993). The protein is a small hydrophobicpeptide of approximately 3200 Da., which can induce an antiviral stateand inhibit both 49S and 26S viral RNA synthesis. Cells treated with theantiviral peptide usually demonstrate quiescent arrest of cellulardivision for 96 hours in uninfected cells, and then normal growth ratesare restored. Cells that have been exposed to this peptide prior toinfection are unable to replicate Sindbis virus and appear to maintainthis phenotype by constitutively producing the antiviral protein through10 months of continuous passage.

It is recognized that this cellular response to Sindbis replication inAedes albopictus cells might decrease the efficiency of a recombinantalphavirus vector producing system in those cells. To improve theefficiency of alphavirus vector production, two methods have beendevised to inactivate the virus-induced cellular antiviral protein, thuspreventing any reduction of vector particle titers. The first methodentails purification of this cellular protein described above, anddetermination of a portion of the primary amino acid sequence usingestablished techniques known in the art. The resulting amino acidsequence is then used to derive possible corresponding genomicsequences, enabling one to design a degenerate PCR primer pair which canbe used to amplify the specific cellular sequence. This amplifiedsequence is then cloned using standard techniques known in the art, toobtain a discreet region of the gene encoding this inhibitory protein.Determination of the nucleotide sequence of this clone then enables oneto design a vector which will integrate specifically within this Sindbisinhibitory gene by homologous recombination, and “knock out” itscapacity to express a functional protein. Cell clones which contain theknock out sequence are identified by insertion of a selectable markerinto the discreet cloned region of the inhibitory protein, prior totransfecting cells with the vector.

A second method for disabling this Sindbis virus inhibitory proteininvolves the treatment of Aedes albopictus-derived packaging cells witha mutagen, for example, BUDR (5-bromodeoxyuridine). The mutagenizedpackaging cell line population is then transfected or transduced with aSindbis vector, which is able to express the neomycin resistance marker.Under high concentrations of the G418 drug, only those cells producinglarge amounts of Sindbis vector, and thus unable to express the Sindbisinhibitory gene, will be able to survive. After selection, resistantcolonies are pooled, dilution cloned, and tested for high titer Sindbisproduction.

2. Modification of Cells to Decrease Susceptibility to AlphavirusExpression: Suppression of Apoptosis and Cytopathology

Packaging cell lines may also be modified by overexpressing the bcl-2gene product in potential parent cell lines, such as canine D-17 andCf2; human HT1080 and 293; quail QT-6; baby hamster kidney BHK-21; mouseneuroblastoma N18; and rat prostatic adenocarcinoma AT-3. The conversionof these cells to a persistently infectable state allows for their useas alphavirus packaging and producer cell lines, similar to those ofretrovector producer lines.

In order to construct such packaging cells, a bcl-2 expression vector isconstructed by using standard recombinant DNA techniques in order toinsert the 910 base pair Eco RI cDNA fragment derived from the plasmidp84 (Nature 336:259) into any commercially available expression vectorcontaining a constitutive promoter and encoding a selectable marker, forexample, pCDNA3 (Introgen, San Diego, Calif.). Careful considerationmust be taken to avoid any type of homology between alphavirus nucleicacid sequences and other transduced vectors. This precaution should betaken in order to prevent recombination events which may lead toundesirable packaging of selectable markers or the bcl-2 oncogene inrecombinant Sindbis particles. This is an important point, since thealphavirus vector system described herein is designed for use as abiological therapeutic. Once the bcl-2 expression vector is constructed,the parent cell line (i.e., BHK-21 cells) is transfected using anystandard technique and selected after 24 hours using the appropriatemarker. Resistant colonies are pooled, followed by dilution cloning, andthen individual clones are propagated and screened for bcl-2 expression.Once expression is verified, persistent Sindbis infection is tested,followed by its use as a parent cell line for alphavirus packaging cellline development.

Other gene products, in addition to the bcl-2 oncogene, which suppressapoptosis may likewise be expressed in an alphavirus packaging orproducer cell line. Three viral genes which are particularly preferredinclude: the adenovirus E1B gene encoding the 19-kD protein (Rao et al.,PNAS 89;7742-7746, 1992), the herpes simplex virus type 1 ₁34.5 gene(Chou and Roizman, PNAS 89:3266-3270, 1992), and the AcMNPV baculovirusp35 gene (Clem et al., Science 254:1388-1390, 1991). These individualgenes may be inserted into any commercially available plasmid expressionvectors, under the control of appropriate constitutive eukaryotictranscriptional promoters, and also containing a selectable marker,using standard techniques. The expression vector constructs aresubsequently transfected into cell lines as described above, and theappropriate selection is applied. Selection for stable integration ofthese genes and constitutive expression their products should allow formore extended vector production in cell lines found to be susceptible toalphavirus-induced apoptotic events. In addition, it is feasible thateach gene product inhibits apoptosis by its own unique mechanism.Therefore, the genes may also be introduced into packaging or producercell lines in various combinations in order to obtain a strongersuppressive effect. Finally, other gene products having similar effectson apoptosis can also be readily incorporated into packaging cell linesas they are identified.

In the deviation of alphavirus vector packaging and producer cell lines,many approaches are outlined to control the expression of viral genes,such that producer cell lines stably transformed with both vector andvector packaging cassettes, can be derived. These approaches includeinducible and/or cellular differentiation sensitive promoters, antisensestructural genes, heterologous control systems, and mosquito or othercells in which viral persistent infections are established. Regardlessof the final configuration for the alphavirus vector producer cell line,the ability to establish persistent infection, or at least delay celldeath as a result of viral gene expression, may be enhanced byinhibiting apoptosis. For example, the DNA tumor viruses, includingadenovirus, HPV, SV40, and mouse polyomavirus (Py), transform cells inpart, by binding to, and inactivating, the retinoblastoma (Rb) geneproduct p105 and its closely related gene product, p107, and other geneproducts involved in the control of the cell cycle including cyclin. Ap33^(cdk2) and p34^(cdc2). All of these viruses, except for Py, encodegene products which bind to and inactivate p53. Uniquely, Py encodesmiddle T antigen (mT) which binds to and activates the membrane tyrosinekinase, src, and also phosphatidy-linositol-3-kinase, which is requiredfor the full transformation potential of this virus (Talmage et al.,Cell 59:55-65, 1989). The binding to and inactivation of the Rb and p53recessive oncogene products prevents cells transformed by these DNAtumor viruses from entering the apoptotic pathway. It is known that p53is able to halt the division of cells, in part by inhibiting theexpression of proteins associated with cellular proliferation, includingc-fos, hsc70, and bcl-2 (Miyashita et al., Cancer Research54:3131-3135,1994).

In order to extend the duration of alphavirus vector production, or topromote a persistently infectable state, packaging and producer cellsare transformed with viral genomic DNA from Py or SV40. In particular,SV40 and Py transformed cell lines are established, and the kinetics andlevel of Sindbis production and cytopathology after viral infectiondetermined. If apoptic events characteristic of Sindbis proliferation inhamster cells are diminished, each prototype alphavirus packaging andproducer cell line subsequently is transformed with Py or SV40, in orderto increase the yield of packaged vector from these cells.

3. Modification of Cells to Decrease Susceptibility of AlphavirusExpression: Production of Activation-Defendent Vector Particles

The Sindbis E2 glycoprotein is synthesized as a precursor, PE2. This PE2precursor along with the second viral glycoprotein, E1, associate in theendoplasmic reticulum and are processed and transported to the infectedcell membrane as a heterodimer for virion incorporation. At some pointduring this processing, PE2 is cleaved into E3 and the mature virionglycoprotein E2, E3 is the 64 amino-terminal residues of PE2 and is lostin the extracellular void during maturation. The larger cleavageproduct, E2, is associated with E1 and anchored in what becomes theviral envelope. Host cell protease(s) is responsible for processing ofthe PE2 precursor, cleaving at a site that immediately follows a highlyconserved canonical four amino acid (aa) residue motif, basic-X-basicaa's. A mutant cell line derived from the CHO-K1 strain, designatedRPE.40 (Watson et al., J. Virol 65:2332-2339, 1991), is defective in theproduction of Sindbis virus strain AR339, through its inability toprocess the PE2 precursor into the E3 and mature E2 forms. The envelopesof Sindbis virions produced in the RPE.40 cell line therefore contain aPE2/E1 heterodimer. RPE.40 cells are at least 100-fold more resistant toSindbis virus infection than the parental CHO-K1 cells, suggesting aninefficiency in the ability of PE2 containing virions to infect thesecells. The defective virions produced by the RPE.40 cell line can beconverted into a fully infectious form by treatment with trypsin.

In packaging and producer cell lines, any wild-type alphavirus that isproduced by recombination between vector and structural protein geneRNAs will re-infect cells and be rapidly amplified, thus, significantlycontaminating and decreasing the tier of packaged vector preparations.Packaging and producer cells developed from the RPE.40 line are analternative to other cell lines permissive for alphavirus infection dueto the inefficient amplification of any wild-type virus generated duringvector production and packaging. Thus, vector preparations are notsignificantly contaminated with wild-type virus. Furthermore, thebenefits of this system are extended to other packaging and producercell lines by developing “knock-out” mutants in their analogous cellularprotease(s), using techniques known in the art.

4. Hopping Cell Line Development

Alphavirus hopping cell lines, as discussed previously, are usedtransiently to produce infectious RNA vector particles which have beenpseudotyped for a different cellular receptor tropism. Once the hoppingcell lines produces vector particles, it is no longer required becauseonly the infectious culture supernatants are needed to transduce theoriginal alphavirus packaging cell lines discussed above. Therefore, thehopping cell line need not exhibit persistent infection by alphavirus inorder to transiently produce vector particles. In this instance, theparent cell line can be either an insect cell line that exhibitspersistent infection, or a mammalian cell line which is likely to lysewithin 24-72 hours after a productive alphavirus infection. The onlycriteria is that the cell lines are able to express either VSV-Gprotein, with or without the appropriate alphavirus structural proteins,or retroviral gag-pol and env protein without affecting cell growthprior to introduction of the alphavirus RNA vector. Therefore, thealphavirus hopping cell line can be any of the aforementioned parentcell lines able to support either alphavirus or retroviral replication,without the additional cell modifications discussed previously, such asbcl-2 oncogene expression.

The generation of VSV-G pseudotyped alphavirus vector particles can beaccomplished by at least three alternative approaches, two of which aredependent on the stable integration of a VSV-G expression cassette intocells. VSV-G protein is known to be highly cytotoxic when expressed incells. Therefore, synthesis of this protein by the expression cassetteis controlled by an inducible promoter. Specifically, a DNA fragmentcontaining the VSV-G protein gene is isolated from plasmid pLGRNL (Emiet al., J. Virol. 65:1202-1207, 1991) by digestion with Bam HI, thetermini made blunt using Klenow fragment enzyme and dNTPs, and the 1.7kb fragment purified from a 1% agarose gel. Plasmid vectorpVGELVIS-SINBV-linker (from Example 3), is digested with enzyme Bsp EIto remove Sindbis nonstructural protein coding sequences nts. 422-7054,and the remaining vector is re-ligated to generate plasmidpVGELVISdlNSP-BV-linker. This plasmid is then digested with Xho I andthe termini made blunt using Klenow fragment enzyme and dNTPs. Thepreviously purified VSV-G fragment is subsequently ligated with thisvector DNA, and resulting clones are screened for proper VSV-G insertorientation. This pVGELVIS-based VSV-G expression construct, in whichVSV-G synthesis is controlled by a Sindbis replicon-inducible junctionregion, is designated pVGELVISdl-G.

Alternatively, a similar Sindbis replicon-inducible VSV-G expressioncassette may be generated in the antisense configuration. In particular,plasmid vector pKSSINBV-linker (described in Example 3) is digested withthe enzymes Apa I and Bam HI to most of the Sindbis nonstructuralprotein coding region, and the resulting 3309 bp vector fragment ispurified from a 1% agarose gel. In addition, plasmid pd5′-26s (describedin section B.3., this example) also is digested with the enzymes Apa Iand Bam HI. The resulting 400 bp fragment which contains the HDVribozyme/Sindbis 5′-end fusion is purified from a 1% agarose gel andsubsequently ligated with the purified pKSSINBV-linker vector fragmentto generate a plasmid designated pd5′-BVlinker. Plasmid pd5′-BVlinker issubsequently digested with Xho I, the termini made blunt using Klenowfragment enzyme and dNTPs, and ligated with the previously purifiedVSV-G fragment. The resulting construct, containing the expressioncassette elements HDV antigenomic ribozyme/Sindbis 5′-end 299nts./Sindbis junction region/VSV-G protein gene/Sindbis 3′-enduntranslated region, is designated as plasmid pd5′-BV-G. Insertion ofthis VSV-G gene cassette into the pcDNA3 vector is as follows. Plasmidpd5′-BV-G is digested with the enzymes Pme I and Apa I, and the terminiare made blunt by the addition of T4 DNA polymerase and dNTPs. Theentire 2.5 kb VSV-G protein gene cassette is purified in a 1% agarosegel. Plasmid pcDNA3 is digested with the enzymes HindIII and Apa I andthe termini are made blunt by the addition of T4 DNA polymerase anddNTPs, and the 5342 bp vector is purified in a 1% agarose gel. The twopurified, blunt-end DNA fragments are subsequently ligated, and theresulting VSV-G protein gene expression cassette vector is known asplasmid pCMV/d5′VSV-G. Further modification of the VSV-G expressioncassettes pVGELVISdl-G and pCMV/d5′VSV-G to substitute other selectablemarkers, for example hygromycin resistance or E. coli gpt, for thecurrent neomycin resistance, or other promoter elements, for exampleDrosophilia metallothionein or hsp 70, for the current CMV, MuLV, andSV40 promoters, may be readily accomplished given the disclosureprovided herein.

In a first VSV-G/alphavirus hopping cell line configuration, VSV-Gexpression cassette plasmid DNA (pVGELVISdl-G or pCMV/d5′VSV-G, ormodified versions thereof is transfected into the appropriate cell type(for example, BHK-21 cells) and selection for G418 resistance is appliedusing media containing 400 g/ml of G418 as described elsewhere in thisexample. G418-resistant cells are cloned by limiting dilution and theindividual cell lines expanded for screening. VSV-G expressing celllines are detected by transfection with any nonstructural proteingene-containing RNA vector (see Example 3) to induce the VSV-Gexpression cassette, followed by immunofluorescence using polyclonalrabbit anti-VSV antibody as described (Rose and Bergmann, Cell34:513-524, 1983). The stably transfected VSV-G expressing cell line, insome cases, is subsequently transfected with plasmid expressioncassette(s) which express one or more Sindbis structural proteins(described elsewhere in this example). For the production of VSV-Gpseudotyped alphavirus particles, the appropriate vector RNA istransfected into the VSV-G hopping cell line, and vectorparticle-containing supernatants are recovered at least 24 hourspost-transfection.

In a second VSV-G/alphavirus hopping cell line configuration, VSV-Gexpression cassette DNA (pVGELVISdl-G or pCMV/d5′VSV-G, or modifiedversions thereof) is transfected into previously derived alphaviruspackaging cell lines (described elsewhere in this example) and theappropriate selection is applied as described previously. The selectedcells are cloned by limiting dilution and the individual cell linesexpanded for screening. VSV-G expressing cell lines are detected bytransfection with any nonstructural protein gene-containing RNA vector(see Example 3) to induce the VSV-G expression cassette, followed byimmunofluorescence using polyclonal rabbit anti-VSV antibody asdescribed (Rose and Bergmann, Cell 34:513-524, 1983). For the productionof VSV-G pseudotyped alphavirus particles, the appropriate vector RNA istransfected into the VSV-G hopping cell line, and vectorparticle-containing supernatants are recovered at least 24 hourspost-transfection.

In a third VSV-G/alphavirus hopping cell line configuration, VSV-Gexpression cassette DNA is co-transfected with the appropriate vectorRNA into previously derived alphavirus packaging cell lines (describedelsewhere in this example). Supernatants containing pseudotyped vectorparticles are recovered at least 24 hours post-transfection.

For the pseudotyping of alphavirus vectors in retroviral packaging celllines, any cell line referenced in the literature, which expressesretroviral gag-pol and env sequences, may be used to package alphavirusRNA vector that has been engineered to contain a retroviral packagingsequence. The retrovirus psi packaging sequence is inserted between theinactivated junction region and a synthetic junction region tandemrepeat, such that only genomic-length vector, and not subgenomic RNA, ispackaged by the retroviral envelope proteins. Retroviral-based particlescontaining alphavirus vector RNA are produced by transfecting in vitrotranscribed alphavirus vector RNA using procedures that have beendescribed previously. Supernatants with pseudotyped retroviral particlescontaining alphavirus RNA vector are harvested at 24 hourspost-transfection, and these supernatants are then used to transduce analphavirus packaging cell line.

5. Identification of Parent Cell Lines Which Produce AlphavirusResistant to Inactivation by Human Complement

Successful intravenous administration of recombinant alphavirusparticles requires that the vector is resistant to inactivation inserum. It is well known to those skilled in the art that Sindbis grownon BHK cells is sensitive to inactivation, in terms of effective virustiter. In order to identify parent cell lines which produce Sindbisparticles which are resistant to inactivation by human complement, thelevel of serum inactivation of Sindbis virus grown on multiple celltypes is tested. The cell types tested are derived from many species,including human, for example, 293 or HT1080 (ATCC No. CCL 121).

As a source of human complement, approximately 70 mls of blood arecollected from patients into serum separating tubes (Becton Dickinson,Los Angeles, Calif.). The blood is allowed to clot for one-half hour atroom temperature. After clotting the serum is centrifuged at 2000 g for10 minutes at 4° C. The serum is collected and placed into a 15 mlconical tube (Corning, Corning, N.Y.) and placed on ice. Approximately,1.1 ml aliquots of the serum are placed in 2 ml cryovials, frozen in adry ice/ethanol bath and stored at −70° C. for subsequent seruminactivation assays. Complement inactivated controls are prepared byheat inactivation of control aliquots for 30 minutes at 56° C.

To test Sindbis for serum inactivation, two vials containing 1.1 ml of100% non-heat inactivated human serum are used for various viruspreparations. One vial of serum is quick thawed at 37° C. The serum isthen heated to 56° C. for 30 minutes to heat inactivate complementpresent in the serum. Following inactivation the serum is placed on ice.The second vial is quick thawed at 37° C. After thawing the serum isplaced on ice.

Approximately, 1.0 ml of the non-heat inactivated serum, medium andheat-inactivated serum are placed in separate 1.5 ml tubes (FisherScientific, Pittsburgh, Pa.) and mixed with 10⁵ Plaque Forming units(PFU) of Sindbis virus and incubated at 37° C. for 1 hour. Afterincubation the tubes are placed on ice.

In order to identify the parent cell line host from which an alphavirusis resistant to human serum inactivation, the non-heat inactivatedserum, medium, and heat-inactivated serum virus preparations are titeredby plaque assay on BHK cells. Equivalent virus titers regardless ofincubation with non-heat inactivation serum, medium, or heat-inactivatedserum, are indicative of parent cell line hosts from which Sindbis virusis resistant to human complement inactivation.

B. Structural Protein Expression Constructs

1. Inducible and Constitutive Structural Protein Vector ExpressionCassettes

The development of alphavirus packaging cell lines is dependent on theability to synthesize high intracellular levels of the necessarystructural proteins: capsid, pE2 and/or E2, and E1. Unfortunately, highlevel expression of these proteins, in particular, the envelopeglycoproteins E2 and E1, may lead to concomitant cytopathology andeventual cell death. Therefore structural protein expression cassetteshave been designed with inducible regulatory elements which control thelevels of gene expression, in addition to others which maintainconstitutive levels of expression.

In a first configuration, expression of the alphavirus structuralproteins is under control of the RSV LTR, in conjunction with theinducible lac operon sequences. This is achieved by insertion ofalphavirus cDNA corresponding to the viral structural protein genes intothe pOP13 and pOPRSV1 vectors (Stratagene). These vectors, usedseparately, are co-transfected with the p3′SS vector (Stratagene), whichexpresses the lac repressor “i” protein. In the absence of inducer, forexample, Isopropyl-B-D-thiogalactopyranoside (IPTG), the basal, orconstitutive, level of expression of a luciferase reporter gene has beenreported to be 10-20 copies per cell. Addition of IPTG, results in aconformational change of the repressor protein, which results indecreased affinity of the lac i protein for lac-operator sequences,permitting high level expression of the heterologous gene. Inductionlevels in the presence of IPTG of 95-fold have been reported forheterologous genes contained in the pOP13 vector.

Specifically, the Sindbis structural protein genes (SP) cDNA is insertedinto the pOP13 and pOPRSV1 vectors as follows. The SP coding region isamplified in toto with a primer pair whose 5′ ends map, respectively, tothe authentic AUG translational start and UGA translational stop sites,including the surrounding nucleotides corresponding to the Kozakconsensus sequence for efficient translational initiation at Sindbis nt7638. The forward primer is complementary to Sindbis nts 7638-7661, andthe reverse primer is complementary to Sindbis nts 11,384-11,364. PCRamplification of Sindbis cDNA corresponding to the structural proteingenes is accomplished by a standard three-temperature cycling protocol,using the following oligonucleotide pair:

Forward primer (7638F):

5′-TATATGCGGCCGCACCACCACCATGAATAGAGGATTCTTTAACATGC-3′  (SEQ. ID NO. 74)

Reverse primer (11384R):

5′-TATATGCGGCCGCTCATCTTCGTGTGCTAAGTCAG-3′  (SEQ. ID NO. 75)

In addition to their respective complementarities to the indicatedSindbis nts, a 5 nucleotide “buffer sequence” followed by the Not Irecognition sequence is attached to the 5′ ends of each primer.Following PCR amplification, the 3,763 bp fragment is purified in a 1%agarose gel, then subsequently digested with the Not I enzyme. Theresulting 3,749 bp fragment is then ligated, separately, into the pOP13and pOPRSV1 vectors, which are digested with Not I and treated with calfintestine alkaline phosphatase. These expression cassette vectors, whichcontain the entire coding capacity of the Sindbis structural proteinsare known as pOP13-SINSP and pOPRSV1-SINSP.

Variations of the lac operon-Sindbis structural protein gene expressioncassettes also can be constructed using other viral, cellular orinsect-based promoters. Using common molecular biology techniques knownin the art, the lac operon and the RSV LTR promoter, or just the RSV LTRpromoter, sequences can be switched out of the Stratagene pOP13 andpOPRSV1 vectors and replaced by other promoter sequences, such as thecytomegalovirus major immediate promoter (pOPCMV-SINSP); the adenovirusmajor late promoter (pOPAMLP-SINSP); the SV40 promoter (pOPSV-SINSP); orinsect promoter sequences, which include the Drosophila metallothioneininducible promoter (pMET-SINSP), Drosophila actin 5C distal promoter(pOPA5C-SINSP), heat shock promoters HSP65 or HSP70 (pHSP-SINSP), or thebaculovirus polyhedrin promoter (pPHED-SINSP).

2. Modification of Cassettes to Increase Protein Expression Levels

Alphavirus structural protein expression can be increased if the levelof mRNA transcripts is increased. Increasing the level of mRNAtranscripts can be accomplished by modifying the expression cassettesuch that alphavirus nonstructural proteins recognize these transcripts,and in turn, replicate the message to higher levels. This modificationis performed by adding the wild-type minimal junction region core(nucleotides 7579 to 7602) to the extreme 5′-end of the Sindbisstructural protein coding region, prior to the first authentic ATG startsite for translation and inverting the expression cassette in thevector, so as to produce antisense structural protein gene transcripts.This can be accomplished by following the same PCR amplificationtechnique described above for placing the Sindbis structural proteincDNA into the pOP13 and pOPRSV1 expression vectors. The onlymodification to this procedure is the replacement of the 7638F forwardprimer with a similar primer that includes junction region corenucleotides 7579-7602 between the Not I restriction enzyme site and thefirst ATG of the coding region as follows:

Forward primer (JUN7638F):

5′-TATATGCGGCCGCATCTCTACGGTGGTCCTAAATAGTACCACCACCATGAATAGAGGATTC-3′  (SEQ.ID NO. 76)

Following PCR amplification, the resulting 3,787 bp fragment is purifiedin a 1% agarose gel, then subsequently digested with the Not I enzyme.The resulting 3,773 bp fragment is then ligated, separately, into thepOP13 and pOPRSV1 vectors which are digested with Not I and treated withcalf intestine alkaline phosphatase. The resulting expression cassettevectors are known as pOP13-JUNSINSP and pOPRSV1-JUNSINSP. However, itmust be stated that the introduction of junction region sequences intothe structural protein expression cassettes will introduce sequenceswhich may possibly lead to undesirable recombination events, leading tothe generation of wild-type virus.

3. Inducible Expression of Structural Proteins via Alphavirus Vector

Because of potential cytotoxic effects from structural proteinexpression, the establishment of inducible packaging cell lines whichexpress even modest basal levels of these proteins may not always bepreferred. Therefore, packaging cell line expression cassettes areconstructed which contain regulatory elements for the high levelinduction of structural proteins synthesis via nonstructural proteinssupplied in trans by the alphavirus vector, but with no basal level ofsynthesis until appropriately stimulated.

In this configuration, a structural protein gene cassette isconstructed, whereby transcription of the structural protein genesoccurs from an adjacent alphavirus junction region sequence. The primaryfeatures of this cassette are: an RNA polymerase II promoter positionalimmediately adjacent to alphavirus nucleotide 1, such that transcriptioninitiation begins with authentic alphavirus nucleotide 1, the 5′-endalphavirus sequences required for transcriptase recognition, thealphavirus junction region sequence for expression of the structuralprotein gene mRNA, the alphavirus structural protein gene sequences, the3′-end alphavirus sequences required for replication, and atranscription termination/polyadenylation sequence. Because of anupstream open-reading frame which ends in transition termination codonsprior to the AUG start site of the structural protein genes, expressionof the alphavirus structural proteins can occur only after the synthesisof minus-strand RNA by vector-supplied nonstructural proteins, followedby the subsequent transcription of a structural protein gene mRNA fromthe junction region. Therefore, the inducibility of this system isdependent entirely on the presence of nonstructural proteins, suppliedby the alphavirus vector itself, introduced as either RNA transcribed invitro, or cDNA positioned downstream of an appropriate promoter element.In addition, the 5′- and 340 -end alphavirus sequences allow for thisRNA transcript of the structural protein gene cassette to be amplifiedby the same vector-supplied nonstructural proteins (see FIG. 11).

Specifically, the construction of a positive-sense, vector-inducibleSindbis packaging cassette is accomplished as follows. Briefly, thepVGEL VIS vector described previously is digested with the enzyme Bsp EIto remove nucleotides 422 to 7054, including most of the nonstructuralgene coding sequences, and the remaining 9925 bp fragment is purified ina 0.8% agarose gel, and subsequently re-ligated to itself to generatethe construct known as pLTR/SindlBspE (FIG. 11). This deletion leavesthe 5′-end authentic translation start codon at nts 60-62 intact, andcreates in-frame downstream UAA and UGA stop codons at nts 7130-7132 and7190-7192 (original numbering), respectively, thus preventingtranslation of the downstream structural protein gene open-readingframe. The pLTR/SindlBspE packaging cassette construct is subsequentlytransfected into BHK cells (ATCC #CCL 10) and transfectants are selectedusing the G418 drug at 400 ug/ml and cloned by limiting dilution. Afterexpansion of the transfected clonal lines, screening for packagingactivity is performed by transfection of Sindbis-luciferase (Sin-luc)vector RNA as described previously. The data shown in FIG. 12demonstrate that transfection of Sin-luc vector RNA into several ofthese clonal LTR/SindlBspE packaging cells results in the production ofinfectious Sindbis particles containing the Sin-luc RNA, as therecovered supernatants are shown to transfer Sin-luc vector RNA to freshmonolayers of BHK cells.

A similar packaging construct can also be made using the pVG-ELVISdclone (described previously) as initial material for creation of the BspEI deletion. In this clone, the Sindbis 3′-end sequence is followed by acatalytic ribozyme sequence to allow more precise processing of theprimary transcript adjacent to the 3′-end sequences of Sindbis. Inaddition, a wide variety of variations of these packaging cassetteconstructions can be made given the disclosure provided herein,including for example, the substitution of other RNA polymerasepromoters for the current MuLV LTR, the addition of 1 or morenucleotides between the RNA polymerase promoter and the first Sindbisnucleotide, the substitution of other ribozyme processing sequences, orthe substitution of a non-Sindbis-encoded open reading frame upstream ofthe structural protein gene sequences, which may or may not retain the5′-end Sindbis sequences required for transcriptase recognition.Furthermore, these constructs can be transfected into other cell lines,as discussed previously.

In another vector-inducible packaging configuration, expressioncassettes contain a cDNA copy of the alphavirus structural protein genesequences flanked by their natural junction and 3′-untranslated regions,and are inserted into an expression vector in an orientation, such thatprimary transcription from the promoter produces antisense structuralprotein gene RNA molecules. Additionally, these constructs contain,adjacent to the junction region, alphavirus 5′-end sequences necessaryfor recognition by the viral transcriptase, and a catalytic ribozymesequence positioned immediately adjacent to alphavirus nucleotide 1 ofthe 5′-end sequence. As such, this ribozyme cleaves the primary RNAtranscript precisely after the first alphavirus nucleotide. In thisantisense orientation, the structural protein genes cannot betranslated, and are dependent entirely on the presence of alphavirusvirus nonstructural proteins for transcription into positive-strandmRNA, prior to their expression. These nonstructural proteins again areprovided by the alphavirus vector itself. In addition, because thisconfiguration contains the precise alphavirus genome 5′- and 3′-endsequences, the structural protein gene transcripts undergo amplificationby utilizing the same nonstructural proteins provided by the alphavirusvector.

Specifically, the Sindbis structural protein gene cDNA is removed fromthe genomic clone pVGSP6GEN and inserted into the pcDNA3 (InvitrogenCorp., San Diego, Calif.) expression vector as follows. First, plasmidpVGSP6GEN is digested with the enzymes Apa I and Bam HI to remove allSindbis sequences through nucleotide 7335, including the genes encodingnonstructural proteins 1, 2, 3, and most of 4. The remaining 7285 bpvector fragment, which contains the Sindbis structural protein genes, ispurified in a 0.8% agarose gel, and subsequently ligated with apolylinker sequence, called SinMCS, that is obtained by annealing twosynthetic oligonucleotides. The oligonucleotides, SinMCSI and SinMCSII,contain the recognition sites for Cla I, Bgl II, and Spe I, and have ApaI and Bam HI ends after annealing. Their sequences are as follows:

SinMCSI:

5′-CTCATCGATCAGATCTGACTAGTTG-3′  (SEQ. ID NO. 77)

SinMCSII:

5′-GATCCAACTAGTCAGATCTGATCGATGAGGGCC-3′  (SEQ. ID NO. 78)

The resulting construct, known as pMCS-26s, is then modified to containthe 5′-end 299 nucleotides of Sindbis, fused to an 84 nucleotideribozyme sequence from the antigenomic strand of hepatitis delta virus(HDV) (Nature 350:434), using overlapping PCR amplification. Two primerpairs are used initially in separate reactions, followed by theiroverlapping synthesis in a second round of PCR. In reaction #1, theforward primer (HDV49-XC) is complementary to HDV genome nucleotides823-859, and the reverse primer (HDV17-68) is complementary to HDVgenome nucleotides 839-887, with sequences as follows:

Forward primer (HDV49-XC):

5′-ACTTATCGATGGTTCTAGACTCCCTTAGCCATCCGAGTGGACGTGCGTCCTCCTTC-3′  (SEQ. IDNO. 79)

Reverse primer (HDV17-68):

5′-TCCACCTCCTCGCGGTCCGACCTGGGCATCCGAAGGAGGACGCACGTCCACT-3′  (SEQ. ID NO.80)

In addition to their respective complementarities, primer HDV49-XCcontains flanking Xba I and Cla I recognition sequences at the 5′-end.PCR amplification of HDV sequences is accomplished by a standardthree-temperature cycling protocol with these primers and Ventpolymerase. In reaction #2, the forward primer (SIN-HDV), which joinsprecisely the HDV and Sindbis sequences, is complementary to nucleotides1-21 of Sindbis, and genomic nucleotides 871-903 of HDV, and overlapsthe sequence of primer HDV17-68 (from above) by 20 nucleotides, and thereverse primer (SIN276-SPE) is complementary to Sindbis nucleotides299-276, with sequences as follows:

Forward primer (SIN-HDV):

 5′-TCGGACCGCGAGGAGGTGGAGATGCCATGCCGACCCATTGACGGCGTAGTACACACT-3′  (SEQ.ID NO. 81)

Reverse primer (SIN276-SPE):

5′-CTGGACTAGTTAATACTGGTGCTCGGAAAACATTCT-3′  (SEQ. ID NO. 82)

In addition to their respective complementarities, primer SIN276-SPEcontains a flanking UAA translation termination codon and SpeIrecognition sequence at its 5′ end. PCR amplification of the fragmentcontaining Sindbis 5′-end sequences fused to HDV ribozyme sequences isaccomplished by a standard three-temperature cycling protocol, usingVent polymerase, these primers, and pVGSP6GEN plasmid as template. Afterthe first round of PCR amplification, {fraction (1/20)}th of the totalamounts from each of reaction #1 and reaction #2 is combined and used astemplate in a second round of PCR amplification with additional input ofprimers HDV49-XC and SIN276-SPE and a standard three-temperature cyclingprotocol. Following the second round of PCR, the 414 bp amplicon ispurified with the MERMAID KIT (Bio101, La Jolla, Calif.), and digestedwith the enzymes ClaI and SpeI. The digested amplicon is purified in a1% agarose gel, and subsequently ligated into plasmid pMCS-26s, whichalso is digested with ClaI and SpeI and purified in a 1% agarose gel.The resulting construct, containing the expression cassette elements HDVantigenomic ribozyme/Sindbis 5′-end 299 nts/Sindbis junctionregion/Sindbis structural protein genes/Sindbis 3′-end untranslatedregion, is known as pd5′26s.

Insertion of the structural protein gene cassette from pd5′26s into thepcDNA3 vector is performed as follows. Plasmid pd5′26s is digested withthe enzyme Xba I and the 3′-recessed ends are made blunt by the additionof Klenow enzyme and dNTPs. The entire 4798 bp structural protein genecassette is purified in a 1% agarose gel. Plasmid pcDNA3 is digestedwith the enzymes HindIII and Apa I and the ends are made blunt by theaddition of T4 DNA polymerase enzyme and dNTPs, and the 5342 bp vectoris purified in a 1% agarose gel. The two purified, blunt-end DNAfragments are subsequently ligated, and the resulting structural proteingene expression cassette vector is known as pCMV-d5′26s (see FIG. 11).Transfection of this DNA into cells and selection for G418 resistance isperformed as previously described.

Modifications of the CMV promoter/antisense-Sindbis structural proteinvector also can be constructed using other viral, cellular, orinsect-based promoters. Using common molecular biology techniques knowin the art, the CMV promoter can be switched out of the InvitrogenpcDNA3 vector and replaced by promoters such as those listed previously.Other variation of this antisense packaging cassette may include, butare not limited to: the addition of 1 or more nucleotides between thefirst Sindbis nucleotide and the catalytic ribozyme, the use of longeror shorter HDV or other catalytic ribozyme sequences for transcriptprocessing, the substitution of a precise transcription terminationsignal for the catalytic ribozyme sequence, or the antisense expressionof structural protein gene cassettes using any downstream sequencerecognized by an RNA polymerase which results in transcription of astructural protein gene mRNA.

Further, it should be noted that each of the vector-inducible constructsdescribed contains sequences homologous to the Sindbis vector itself.Therefore, the potential exists for the generation of wild-type virus byrecombination between the two RNA molecules. Additional modificationsmay be made to eliminate this possibility as described below.

4. Separation of Structural Protein Genes to Prevent Recombination

Packaging cell lines may also be generated which segregate theintegration and expression of the structural protein genes, allowing fortheir transcription as non-overlapping, independent RNA molecules. Forexample, the expression of capsid protein independently of glycoproteinsE2 and E1, or each of the three proteins independent of each other,eliminates the possibility of recombination with vector RNA andsubsequent generation of contaminating wild-type virus.

Specifically, capsid protein is expressed independently from aninducible expression vector, such that sequences which might result inrecombination with vector RNA are eliminated. As an example, the capsidprotein gene is amplified from plasmid pVGSP6GEN with a primer paircomplementary to nucleotides 7632-7655 (forward primer) and 8415-8439(reverse primer), with sequences as follows:

Forward primer (Sin7632F):

5′-GTCAAGCTTGCTAGCTACAACACCACCACCATGAATAGAG-3′  (SEQ. ID NO. 83)

Reverse primer (Sin8439R):

5′-CAGTCTCGAGTTACTACCACTCTTCTGTCCCTTCCGGGGT-3′  (SEQ. ID NO. 84)

In addition to their respective complementarities, the forward primercontains Nhe I and HindIII recognition sequences at its 5′-end, and thereverse primer contains both UAG and UAA translation stop codons and aXho I recognition sequence at its 5′-end. Amplification is accomplishedusing a standard three-temperature cycling protocol, and the resultingamplicon is digested with the enzymes Nhe I and Xho I, and purified in a1% agarose gel. Expression plasmid pMAM (Clontech), which contains adexamethasone-inducible MMTV LTR promoter sequence, is digested with theenzymes Nhe I and Xho I and the plasmid DNA purified in a 1% agarosegel. The capsid protein gene fragment is ligated into the pMAM vector,and the resulting construct is known as pMAM/C. Plasmid pMAM/C istransfected into the appropriate cell line (for example BHK-21) asdescribed previously and selection for stable transfectants isaccomplished by using HAT (hypoxanthine, aminopterin, thymidine) media,supplemented with dialyzed fetal calf serum, mycophenolic acid andxanthine, as described by Mulligan and Berg (PNAS 78:2072-2076, 1981).HAT-selected cell lines expressing capsid protein are identifiedfollowing induction with 10⁻⁶ M dexamethasone by lysing the cells withLammeli sample buffer, separating the proteins using 12% SDS-PAGE,blotting onto nitrocellulose membrane, and detecting by western blotusing polyclonal rabbit anti-Sindbis antibody. FIG. 21 shows expressionof capsid protein in such cells, along with wild-type BHK-21 cells as anegative control, and Sindbis virus-infected BHK-21 cells as a positivecontrol.

Alternatively, capsid protein is expressed using the lac-induciblevectors (Stratagene) described previously. The Sindbis capsid proteingene is amplified by PCR using primers Sin7632F and Sin8439R (describedpreviously), and ligated with TA vector DNA (Stratagene). The resultingplasmid, designated TA/SinC, is digested with Eco RI, the termini aremade blunt by the addition of Klenow fragment enzyme and dNTPs, and thecapsid protein gene purified from a 1% agarose gel. Plasmid vectorspOP13 and pORSV1 are digested with Not I, their termini made blunt bythe addition of Klenow fragment enzyme and dNTPs, and subsequentlytreated with calf intestinal alkaline phosphatase. The capsid proteingene is ligated with both pOP13 and pORSV1 vector DNA to generate theexpression constructs designated pOP13CAP and pORSV1CAP, respectively.Each plasmid is co-transfected with p3′SS into the appropriate cell lineas described previously, and selection for stable transfectants isaccomplished using G418 and hygromycin selection. Cell lines expressingcapsid protein are identified following IPTG induction byimmunofluorescence using polyclonal rabbit anti-Sindbis antibody.

The glycoprotein genes, E1 and E2, are expressed together using one ofthe inducible systems previously described. For example, the Sindbis E1and E2 genes are amplified from plasmid pVGSP6GEN using a primer paircomplementary to Sindbis nucleotides 8440-8459 (forward primer) andSindbis nts 11,384-11,364 (reverse primer). PCR amplification isperformed using a standard three-temperature cycling protocol and thefollowing oligonucleotide pair:

Reverse primer (11384R):

5′-TATATGCGGCCGCTCATCTTCGTGTGCTAGTCAG-3′  (SEQ. ID NO. 75)

Forward primer (8440F):

5′-TATATGCGGCCGCACCACCATGTCCGCAGCACCACTGGTCACG-3′  (SEQ. ID NO. 85)

In addition to their respective complementarities, the forward primercontains an “in-frame” AUG translation initiation codon, and bothprimers contain a NotI recognition sequence at their 5′-ends. FollowingPCR amplification, the amplicon is digested with the NotI enzyme andpurified in a 1% agarose gel. The resulting fragment is then ligatedseparately into the pOP13 and pOPRSV1 vectors (Stratagene), digestedwith Not I and treated with calf intestinal alkaline phosphatase, asdescribed previously. These glycoprotein expression vectors are used totransfect cells that have been previously transfected with a capsidprotein expression construct, and stable glycoprotein gene transfectantsare identified by selection for G418 and hygromycin resistance.

Alternatively, the E1 and E2 glycoproteins are expressed under thecontrol of the replicon-inducible junction region promoter, describedpreviously. The ELVIS expression plasmid pVGELVIS0SINBV-liner (Example3) is digested with the enzyme Not I, and treated with calf intestinalalkaline phosphatase. PCR amplified Sindbis E1 and E2 glycoprotein genesdigested with Not I (previous paragraph) are then ligated to the ELVISvector to generate a construct designated pVGELVIS-E1/E2. PlasmidpVGELVIS-E1/E2 subsequently is digested with the enzyme Bsp EI, removingmost of the nonstructural protein gene coding region, and the remainingE1- and E2-containing vector DNA is re-ligated to itself, creating aninducible expression cassette identified as pVGELVdl-E1/E2. Thisglycoprotein expression vector is used to transfect cells that have beenpreviously transfected with a capsid protein expression construct, andstable glycoprotein gene transfectants are identified by selection forG418 resistance. For both the capsid and envelope glycoproteinexpression cassettes, additional mammalian or non-mammalian (includinginsect)-derived promoters, which may or may not be inducible, arereadily substituted for those described above, using standard techniquesknown in the art.

5. Assembling the Components to Create the Alphavirus Packaging CellLine

For example purposes, the BHK-21 cell line and replicon-induciblepackaging expression cassette are used to demonstrate assembly of thecomponents. However, other possible parent cell lines can be used tocreate alphavirus packaging cell lines and have been discussedpreviously. Briefly, BHK-21 cells (CCL 10) are grown at 37° C. in 5% CO₂in Dulbecco's modified Eagle's Media (DMEM), 2 mM L-glutamine, and 10%fetal bovine serum (optimal media). Approximately 5×10⁵ BHK-21 cells,grown in a 35 mM petri dish, are transfected with 5 ug pLTR/SindIBspEusing 5 ul of the Transfectam (Promega) cationic lipid reagent, inserum-free media conditions, as suggested by the supplier. However, anymethod of transfection is rapidly substituted, i.e., by electroporation,calcium phosphate precipitation, or by using any of the readilyavailable cationic liposome formulations and procedures commonly knownin the art. At 24 hours post-transfection, the cells are trypsinized andreseeded in 100 mm dishes in 10 ml of optimal media, as described above,supplemented with 400 ug/ml of G418 (Gibco/BRL) and selected over aperiod of 5 to 7 days. Colonies displaying resistance to the G418 drugare then pooled, dilution cloned, and propagated. Individual clones arescreened for high levels of Sindbis structural protein expression andfunctional packaging after transfection with Sindbis-luciferase vectorRNA transcribed in vitro from SacI linearized plasmid pKSSINBV-luc (seeExample 3). Specifically, clonally-derived pLTR/SindlBspE transfectedBHK-21 cells (referred to as LTR/SindlBspE or BK-Bsp cells) grown in 60mm petri dishes are transfected with 2 ug of Sindbis-luciferase vectorRNA and overlayed with 3 ml of optimal media (see above). At 20 hourspost-transfection, the supernatants are removed, and clarified bycentrifugation for 30 min. at 3000 rpm in a Sorvall RT6000B tabletopcentrifuge. In addition, the transfected cell monolayer is lysed inreporter lysis buffer (Promega) as described by the manufacturer, andassayed for luciferase expression as described previously.

The transfer of luciferase activity (and thus functional packaging) istested by using 1 ml of the above supernatants to infect freshmonolayers of BHK-21 cells in 60 mm dishes. At 20 hours post-infection,the cell monolayers are lysed as described above, and tested forluciferase expression. As shown in FIG. 12, three clones (#13, 18, and40) produce packaged Sindbis-luciferase vector and are the firstexamples of alphavirus packaging cell lines. In addition, transfectedclone #18 cells are tested for increased vector packaging over atimecourse following transfection. Supernatants from transfected clone#18 cells are harvested at 20, 45, and 70 hours post-transfection, asdescribed above, and used to infect fresh monolayers of BHK-21 cells.FIG. 13 shows that Sindbis-luciferase vector packaging increasessignificantly at 45 hours post-transfection, as compared to 20 hourspost-transfection. Expression also can be tested by western blotanalysis using polyclonal rabbit anti-Sindbis antibodies (available inthe literature).

C. INDUCIBLE VECTOR AND STRUCTURAL PROTEIN EXPRESSION FOR ALPHAVIRUSPRODUCER CELL LINES

1. Use of Viral Promoters

The challenge of developing an alphavirus vector producer cell line liesin the question of whether a virus, whose infection of mammalian cellsresults almost exclusively in productive lytic cell death, can bemodified to establish persistent infection in these same cells. Oneapproach is to generate alphavirus vector producer lines from mosquitocells, where viral persistence often results after infection. However,the titer of infectious virus produced in persistently infected mosquitocells is only about 1×10⁴ PFU/ml, at least five orders of magnitude lessthan that observed after lytic infection of BHK cells by Sindbis.

Several strategies are described for inducible alphavirus vectorproducer cell lines, containing both vector and viral structural genecassettes, such that productive cytolytic infection occurs only afterthe correct stimulus. Because these approaches operate on a “feedforward” level, any leakiness in the system will result in initiation ofthe alphavirus replication cycle and probable cell death. Therefore,tightly regulated control mechanisms are necessary for such a system.

The hallmark of development in the differentiation state-dependentpattern of gene expression. Briefly, gene expression patterns differwidely between undifferentiated and terminally differentiated states.Thus, a cell whose differentiation state can be controlled is likely anideal host in which to derive an alphavirus vector producer cell line.In such a configuration, the vector expression cassette and, in someinstances, structural components are coupled to terminal differentiationstate-inducible promoters, according to the strategy described forELVIS, and used to transform stably an undifferentiated host cell.Terminal differentiation of the host producer cell after induction withthe appropriate stimuli coincidentally results in induction of thealphavirus replication cycle and production of packaged vector. Otherstrategies described herein, including antisense structural genes andheterologous viral expression systems, are readily coupled with cellulardifferentiation state-dependent promoters described below.

In this approach, four examples are described, using either a viral orcellular promoter which are active in only terminally differentiatedcells.

It has been shown that mouse Polyomavirus (Py), SV40, and Moloney murineleukemia virus (M-MuLV), all are able to infect and enterundifferentiated mouse embryonal carcinoma (EC) cells, but theexpression of their genes (and heterologous genes) and establishment ofproductive infection is blocked (Swartzendruber and Lehman, J. Cell.Physiol. 85:179-188, 1975; Peries et al., J. Natl. Cancer Inst.59:463-465, 1977). These viral growth properties also have beendemonstrated in two cell lines, PCC4 and F9, which are derived from themalignant stem cells of mouse teratorcarcinomas. The block to viralpropagation occurs at the level of transcription and replication, andmaps to the enhancers, contained within the viral non-coding controlregions (Linney et al., Nature 308:470-472, 1984; Fujimura et al., Cell23:809-814, 1981; Katinka and Yaniv, Cell 20:393-399, 1980). When M-MuLVinfects undifferentiated EC cells, the viral DNA integrates into thegenome. However, as stated above, expression of viral genes or ofheterologous genes is blocked. This block of viral expression isreleased upon terminal differentiation of EC cells by addition ofretinoic acid to the growth medium.

To test the RNA expression properties of the pVGELVIS construct in ECcells, plasmid DNA is complexed with LIPOFECTAMINE (GIBCO-BRL,Gaithersburg, Md.) according to the conditions suggested by the supplier(ca. 5 g DNA/8 g lipid reagent) and added to 35 mm wells containingundifferentiated PCC4 or F9 cells (Fujimura et al., 1981, Cell23:809-814) at approximately 75% confluency. The development ofcytopathic effects (CPE), and the level of Sindbis productive infection,quantitated by plaque assay of media supernatant, is determined atregular intervals over 5 days in undifferentiated and differentiatedtransfected PCC4 or F9 cells. Differentiation of F9 and PCC4 cells isaccomplished by addition of retinoic acid (Sigma Chemical Co., St.Louis, Mo.), at a final concentration of 1 M.

It has been proposed that the hierarchy of relative expression ofheterologous genes observed in undifferentiated EC cells infected withM-MuLV vectors may be in part insertional dependent (Linney et al.,1987, J. Virol. 61:3248-3253). Thus, undifferentiated EC cellstransfected with pVGELVIS may likely produce different results, in termsof transcription of the Sindbis genomic cDNA and, in turn, initiation ofthe viral life cycle. In this event, following G418 selection ofpVGELVIS transfected undifferentiated EC cells, remaining cells arecloned and expanded. The cell clones are then tested for the productionof Sindbis virus after differentiation by addition of retinoic acid(Sigma Chemical Co., St. Louis, Mo.), at a final concentration of 1 M.

To isolate vector packaging cell lines, whose production of structuralproteins in the presence of Sindbis NSP is cell differentiation statedependent, undifferentiated F9 or PCC4 cells are transfected withpLTR/SINdlBspE and G418 selected as described above. Differentiationstate-sensitive clones are then selected by infection at highmultiplicity with packaged SIN-luc vector. Clones which are resistant tocell lysis or do not produce packaged SIN-luc vector particles, arecandidate vector packaging clones. These candidate clones are tested forSIN-luc vector particle production following terminal differentiationwith retinoic acid, as described.

The murine wild type polyomavirus (Py) is unable to replicate in theteratocarcinoma cell lines PCC4 or F9. This block of replication inundifferentiated cells occurs at the level of transcription of earlyregion (i.e., T antigen) genes, and is released by induction of terminaldifferentiation with vitamin A. Py mutants which are able to establishproductive infection in undifferentiated PCC4 and F9 cells map to theviral enhancer region. The genesis of an embryonic tissue specifictranscriptional enhancer element has resulted in these mutants. In orderto exploit this property of inhibition of Py replication inundifferentiated teratocarcinoma cell lines, the viral regulatorynon-coding region, including the enhancer, is coupled to the genomiccDNA of Sindbis virus, according to the ELVIS strategy. The precisetranscriptional start site of the Py early region has been determined(see Tooze, DNA Tumor Viruses). The PCC4 and F9 cell lines are stablytransformed with the Py-Sindbis vectors. In this model Sindbisproductive infection occurs after addition of retinoic acid to theculture medium and induction of terminal differentiation.

The Py non-coding region from bases 5021-152, which includes thesequences corresponding to the viral enhancers, 21 bp repeats,replication origin, CAAT and TATA boxes, and the early mRNAtranscription 5′ cap site, is positioned at the 5′ viral end such that nvivo, only a single capped C residue is added to the Sindbis 5′ end.Juxtaposition of the Py non-coding region and the Sindbis 5′ end isaccomplished by overlapping PCR as described in the following detail.Amplification of the Py non-coding region in the first primary PCRreaction is accomplished in a reaction containing the pBR322/Py, strainA2 plasmid (ATCC number 45017-p53.A6.6(pPy-1)) and the following primerpair:

Forward primer: Pybgl5021F (buffer sequence/Bgl II recognitionsequence/Py nts 5021-5043):

5′-TATATAGATCTCTTGATCAGCTTCAGAAGATGGC (SEQ. ID NO. 86)

Reverse primer: SINPy152R (SIN nts 5-1/Py nts 152-134):

5′-TCAATGGCGGGAAGAGGCGGTTGG (SEQ. ID NO. 87)

PCR amplification of the Py non-coding region with the primer pair shownabove is performed using the Thermalase thermostable DNA polymerase(Ameresco Inc., Solon, Ohio) and the buffer containing 1.5 mM MgCl₂,provided by the supplier. Additionally, the reaction contains 5% DMSO,and the Hot Start Wax beads (Perkin-Elmer), using the following PCRamplification protocol shown below:

Temperature (° C.) Time (Min.) No. Cycles 94 2 1 94 0.5 55 0.5 35 72 0.572 10 1

Amplification of the Sindbis 5′ end in the second primary PCR reactionis accomplished in a reaction containing the pVGSP6GEN clone and thefollowing primer pair:

Forward primer: (Py nts 138-152/SIN nts 1-16):

5′-CCGCCTCTTCCCGCCATTGACGGCGTAGTAC (SEQ. ID NO. 88)

Reverse primer: (SIN nts 3182-3160):

5′-CTGGCAACCGGTAAGTACGATAC (SEQ. ID NO. 18)

PCR amplification of Sindbis 5′ end region with the primer pair shownabove is with the reaction conditions described above, using thefollowing PCR amplification protocol shown below:

Temperature (° C.) Time (Min.) No. Cycles 94 2 1 94 0.5 55 0.5 35 72 3.072 10 1

The 442 bp and 3202 bp products from the primary PCR reactions arepurified with GENECLEAN (BIO 101), and used together in a PCR reactionwith the following primer pair:

Forward primer: Pybgl5021F (buffer sequence/Bgl II recognitionsequence/Py nts 5021-5043):

5′-TATATAGATCTCTTGATCAGCTTCAGAAGATGGC (SEQ. ID NO. 89)

Reverse primer: (SIN nts 2300-2278):

 5′-GGTAACAAGATCTCGTGCCGTG (SEQ. ID NO. 19)

PCR amplification of the of the primer PCR amplicon products with theprimer pair shown above is with the reaction conditions described above,using the following PCR amplification protocol shown below:

Temperature (° C.) Time (Min.) No. Cycles 94 2 1 94 0.5 55 0.5 35 72 3.072 10 1

The 20 3′ terminal bases of the first primary PCR amplicon productoverlaps with the 20 5′ terminal bases of the second primary PCRamplicon product; the resultant 2,742 bp overlapping secondary PCRamplicon product is purified by 0.8% agarose/TBE electrophoresis,digested with Bgl II, and the 2,734 bp product is ligated intopcDNASINbgl/xba (see Example 3) treated with Bgl II and CIAP. Theresulting construction is 16,641 bps and is known as ELVIS-PySIN. Inorder to construct a structural protein expression vector similar topLTR/SindlBsp for the derivation of vector packaging cell lines, theELVIS-PySIN construction is digested to completion with Bsp EI, andreligated under dilute conditions, in order to accomplish deletion ofthe nonstructural proteins between bases 422-7054. This construction isknown as ELVIS-PySINdlBspE.

ELVIS-PySIN plasmid DNA is complexed with LIPOFECTAMINE (GIBCO-BRL,Gaithersburg, Md.) according to the conditions suggested by the supplier(ca. 5 g DNA/8 g lipid reagent) and added to 35 mm wells containingundifferentiated PCC4 or F9 cells at approximately 75% confluency. Thedevelopment of cytopathic effects (CPE), and the level of Sindbisproductive infection, quantitated by plaque assay of media supernatant,is determined at regular intervals of 5 days in undifferentiated anddifferentiated PCC4 or F9 cells. Differentiation of F9 and PCC4 cells isaccomplished by addition of retinoic acid (Sigma Chemical Co., St.Louis, Mo.), at a final concentration of 1 mM.

If the undifferentiated EC cells demonstrate a heterologous response totransfection with ELVIS-PySIN, remaining cells not lysed by Sindbisvirus propagation following G418 selection of pVGELVIS transfectedundifferentiated EC cells are cloned and expanded. The cell clones arethen tested for the production of Sindbis virus after differentiation,by addition of retinoic acid (Sigma Chemical Co., St. Louis, Mo.), at afinal concentration of 1 mM.

Isolation of vector packaging cell lines stably transfected withELVIS-PySINdlBspE, having a cell differentiation state dependent patternof expression of structural proteins in the presence of Sindbis NSP, isaccomplished as described above for the pLTR/SindlBspE plasmid.

In order to demonstrate the feasibility of an inducible Sindbis vectorproducer cell line, the reporter gene expression from the ELVIS-lucvector, whose construction is described in Example 3, section E, aftertransfection of BHK and undifferentiated F9 cells is determined. Inaddition, both cell types are infected with packaged SIN-luc vector,whose production is described in Example 3 section C. This laterexperimental group serves as a control that expression restriction (ifany) lies at the level of transcription rather than a receptordifference on unique cell types. The results of this study, shown inFIG. 14, demonstrate that the expression of luciferase is inhibited inundifferentiated F9 cells. The level of luciferase expression in BHKcells transfected with ELVIS-luc and BHK and undifferentiated F9 cellsinfected with packaged SIN-luc vector is similar. Thus, in ELVIS-luctransfected undifferentiated F9 cells, transcription from the LTR andsubsequent luciferase expression via the Sindbis vector autocatalyticpathway is inhibited. This study demonstrates that packaging cell linescan be developed where synthesis of Sindbis vector or Sindbis vectorpackaging is inducible and controlled by the differentiation state ofthe cell.

2. Use of Cellular Promoters

The third example of this strategy uses the β-globin locus controlregion. The β-globin multigene cluster contains five developmentallyregulated genes. In the early stages of human development, the embryonicyolk sac is the hematopoietic tissue and expresses the ε-globin gene.This is followed by a switch to the γ-globin gene in the fetal liver andthe δ- and β-globin genes in adult bone marrow (Collins and Weissman,1984, Prog. Nucleic Acid Res. Mol. Biol. 31:315).

At least two mouse erythroleukemia lines, MEL and Friend, serve asmodels for terminal differentiation dependent expression of β-globin.Expression of β-globin is observed in these lines only after inductionof terminal differentiation by addition of 2% DMSO to the growth medium.

The entire β-globin locus is regulated by the locus control region(LCR). Within the LCR is the dominant control region (DCR) residingwithin the DNase I hypersensitive region, which is 5′ of the codingregion. The DCR contains five DNase I hypersensitive (HS1-HS5) sites.The DCR directs high level site of integration independent, copy numberdependent expression on a linked human β-globin gene in transgenic miceand stably transfected mouse erythroleukemia (MEL) cells (Grosveld etal., 1993, CSHSQB 58:7-12). In a recent study (Ellis et al., 1993, EMBO12:127-134), concatamers of a synthetic core coinciding to sequenceswithin HS2 were shown to function as a locus control region.

In order to accomplish the differentiation state dependent expression ofalphavirus vectors, the viral genomic cDNA is juxtaposed with a promotercontaining a tandem synthetic core corresponding to the LCR HS2 site.Alternatively, the desired alphavirus vector construct can be inserteddownstream of the LCR in the endogenous -globin gene by homologousrecombination. In such a strategy, the β-globin transcription initiationsite after terminal differentiation would be first determined, in orderthat the alphavirus vector could be placed precisely at the start site.

Initiation of a lytic viral life cycle is controlled by thedifferentiation state of the host cell is applicable to other systems,where the control of viral induced cytopathology is desired.

Yet another approach to regulating alphavirus gene expression through adifferentiation state sensitive promoter is the use of the retinoic acidreceptor a (RARA) and acute promyelomonocytic leukemia cells (APL). APLcells are clonal myeloid precursors characterized by high growth rateand differentiation arrest. A non-random chromosomal translocationbreakpoint, t(15;17)(q22;21), occurs in almost all patients with APL.The RARA gene has been localized to chromosome 17q21. Analysis of APLmRNA from patients has shown that most APL breakpoints occur within thesecond intron of the RARA gene and result in abnormal fusiontranscripts. Co-transfection assays with RARA and PML-RARA fusion cDNAshave demonstrated that the resulting fusion proteins can antagonizewild-type RARA in the presence of the retinoic acid. These studiesimplicate PML-RARA fusion protein in the molecular pathogenesis of APL.Importantly, a significant number of patients achieve complete remissionafter all-trans retinoic acid treatment (ATRA). High concentration ofATRA may overcome the RARA deficiency leading to high levels of RA inthe nucleus. Differentiation of the APL cells can then be achievedthrough activation of RARA responsive genes. RA can inducedifferentiation of a number of cell lines, including the human leukemialine HL-60.

The retinoic acid receptor is a member of a nuclear receptor superfamilythat includes the thyroid and steroid hormone receptors. Four differentforms of the human RAR have been identified, and the corresponding cDNAscloned and characterized. In order to accomplish the differentiationstate dependent expression of Sindbis vectors, viral genomic cDNA isjuxtaposed with the RARA DNA binding site, creating ELVIS-RARASIN. Aswith the strategy proposed for ELVIS-PySIN expression inundifferentiated EC cells, differentiation sensitive ELVIS-RARASINexpression cells are isolated.

3. Insertion of Vector Constructs into Differentiation State ControlledInducible Promoters

Generation of clones whose expression of heterologous genes from Sindbisvectors positioned in the ELVIS configuration as described in Example 3is differentiation state dependent, is accomplished as described abovefor the pVGELVIS, pLTR/SindlBspE plasmids. Generation of clones whoseproduction of vector particles is differentiation state dependent, isaccomplished by transfecting the isolated differentiation dependentvector packaging clones described above with ELVIS heterologous geneexpression vectors. Clones having the desired phenotype or vectorproduction after retinoic acid induced differentiation are isolated asdescribed above.

D. STRUCTURAL PROTEIN EXPRESSION FROM A HETEROLOGOUS ASTROVIRUS JUNCTIONREGION

Among the critical properties of a vector packaging system are a cellline which expresses the structural components necessary to generate aninfectious particle, without the creation of wild-type virus throughrecombination between vector and structural gene components. These twodesired properties of the packaging cell line are accomplished in theretrovirus based systems through the constitutive expression of thegag/pol and env genes on individual heterologous RNA polymerase IIexpression cassettes.

Another important aspect of vector packaging cell lines is to derive asystem which mimics as closely as possible the normal replicationstrategy of the wild type virus. This issue is important in terms of theobserved titer level of packaged recombinant vector. Synthesis of theviral structural proteins during alphavirus infection is accomplishedafter transcription of high levels of subgenomic mRNA from the junctionregion promoter, followed by efficient translation into the structuralproteins. The junction region promoter is functional only in theantisense orientation and synthesis of the antigenomic RNA occurs aftertranslation of the nonstructural proteins, thus delaying the expressionof the structural proteins. It follows that, with regard to alphavirus,it would be desirable to construct a packaging cell line in whichsynthesis of the structural proteins is initiated from the junctionregion promoter, which in turn is activated by nonstructural proteinsexpressed from the recombinant vector molecule.

It is known that a relatively high frequency of recombination occursbetween RNA genomic molecules occurs during infection with Sindbis virusvia a copy choice mechanism (PNAS 88:3253-3257, 1991). Recombinationbetween vector and junction region/structural gene cassettes wouldresult in the generation of wild-type Sindbis virus, perhaps at a levelof 1 wild-type virus per million of packaged vector particles(Liljestrom Bio Technology 9:1356-1361, 1991). One way to mitigate thegeneration of wild-type virus is to separate the structural genes ontoseparate expression cassettes, an approach which has been discussedpreviously in Example 7.

An additional approach to diminish the level of wild-type virusproduction in alphavirus vector packaging cell lines is to express thestructural proteins under the control of Astrovirus genetic elements. Aschematic for this configuration is depicted in FIG. 15. Similar toalphaviruses, the expression of Astrovirus structural proteinsincorporates a junction region strategy, in which high levels ofstructural proteins are synthesized from a subgenomic message. TheAstrovirus expression cassette may consist of one of the two followingordered elements: (1) inducible promoter/Astrovirus 5′ end/Astrovirusjunction region/alphavirus structural gene/Astrovirus 3′ end, or (2)antisense Astrovirus 3′ end/antisense alphavirus structuralgene/antisense Astrovirus junction region/antisense Astrovirus 5′end/Hepatitis Delta virus ribozyme, or other configurations described inExample 7. In both configurations, the expression unit is amplified bythe Astrovirus nonstructural proteins through the same mechanism thatoccurs during viral replication. Since multiple rounds of subgenomicmRNA synthesis initiated from the junction region occur from eachexpression unit, amplification of the expression unit by the Astrovirusnonstructural proteins results in the production of very high levels ofalphavirus structural proteins. The second configuration of thealphavirus structural protein expression cassette described above mayfunction better than the first, because the primary transcript of thetoxic alphavirus structural gene is antisense. Although expression ofthe structural genes in the first configuration should not occur untilsynthesis of the negative strand followed by synthesis of the positivesubgenomic RNA from the junction region, the antisense nature of theprimary transcript in the second configuration represents an additionallevel of control to prevent cytotoxic protein expression.

It is likely that no wild-type virus would be generated in a packagingcell line in which the alphavirus virus structural proteins aresynthesized individually from Astrovirus junction region expressioncassettes. Recombination between the nonstructural protein region of thevector and an Astrovirus structural protein expression cassette wouldresult in a molecule in which Astrovirus cis elements were coupled withalphavirus genes, a nonviable combination. Correct coupling ofalphavirus cis and trans elements would require two preciserecombination events between the vector and the Astrovirus expressioncassette, between the Astrovirus junction region and structural geneATG, and between the structural gene termination codon and theAstrovirus 3′ end. In order to generate wild type virus, this dualrecombination event would have to occur three times on the same molecule(six total events), to incorporate the three separated alphavirusstructural genes.

In order to diminish any possible toxicity of the Astrovirus proteins,synthesis of the Astrovirus expression cassettes may also be controlledby inducible promoters. One possibility is to use the lac operon,according to the “lac-switch” system described previously in Example 7(Stratagene). The constitutive level of expression of the lac operoncontrolled gene in the absence of the gratuitous inducer IPTG is about10 copies of RNA per cell. The inducible promoter corresponding to theAstrovirus/alphavirus structural gene expression cassette may be the lacoperon or other suitable promoters which have very low level ofconstitutive expression. Construction of packaging cell lines of theseconfigurations, in which the control of alphavirus proteins is directedby a heterologous virus should result in the generation of high titerwild-type virus free packaged vector particles.

Example 8 Alternative Viral Vector Packaging Techniques

Various alternative systems can be used to produce recombinantalphavirus particles carrying the vector construct. Each of thesesystems takes advantage of the fact that baculovirus, and the mammalianviruses vaccinia and adenovirus, among others, have been adaptedrecently to make large amounts of any given protein for which the genehas been cloned. (Smith et al., Mol. Cell. Biol. 3:12, 1983; Piccini etal., Meth. Enzymology 153:545, 1987; and Mansour et al., Proc. Natl.Acad. Sci. USA 82:1359, 1985). These and other viral vectors are used toproduce proteins in tissue culture cells by insertion of appropriategenes into the viral vector and can be readily adapted to makealphavirus vector particles.

For example, adenovirus vectors are derived from nuclear replicatingviruses and can be modified so they are defective. Heterologous genesare inserted into these vectors either by in vitro construction (Ballayet al., EMBO J. 4:3861, 1985) or by recombination in cells (Thummel etal., J. Mol. Appl. Genetics 1:435, 1982), and used to express proteinsin mammalian cells. One preferred method is to construct plasmids usingthe adenovirus major late promoter (MLP) driving: (1) alphavirusstructural proteins; and (2) an alphavirus vector construct. Thealphavirus vector in this configuration still contains a modifiedjunction region, and would allow the transcribed RNA vector to beself-replicating, as in previously described configurations.

These plasmids are then used to make adenovirus genomes in vitro (Ballayet al., EMBO. J. 4:3861, 1985). The recombinant adenoviral genomes,which are replication defective, are separately transfected into 293cells (ATCC #CRL 1573, a human cell line making adenovirus E1A protein),to yield pure stocks of defective adenovirus vectors expressing eitheralphavirus structural proteins or alphavirus vectors. Since the titresof such vectors are typically 10⁷-10¹¹/ml, these stocks are then used toinfect tissue culture cells simultaneously at high multiplicity ofinfection, resulting in the production of alphavirus proteins and vectorgenomes at high levels. Since the adenovirus vectors are defective,little or no direct cell lysis will occur and vectors are harvested fromthe cell supernatants. Similar approaches are readily carried out usingrecombinant vaccinia virus vectors constructed by inserting thealphavirus sequences into the shuttle plasmid pK (Bergmann et al., Eur.J. Immunol. 23:2777, 1993) for in vivo recombination into the vacciniaWR strain.

Other viral vectors, such as those derived from unrelated vectors (e.g.,RSV, MMTV or HIV), also may be used in the same manner to generatepackaged vectors from primary cells. In one embodiment, these adenoviralvectors are used in conjunction with primary cells, giving rise torecombinant alphavirus particles.

An alternative expression system also has been described in whichchimeric HIV/poliovirus genomes result in the generation of chimericminireplicons (J. Virol. 65:2875, 1991) capable of expressing fusionproteins. These chimeric poliovirus minireplicons, in which HIV-1gag-pol sequences were substituted for the VP2 and VP3 capsid genes ofthe P1 capsid of poliovirus, were later demonstrated to be encapsidatedand produce infectious particles by using a recombinant vaccinia virus(VV-P1) that expresses the substituted poliovirus capsid precursor P1proteins defective in the chimeric minireplicon (J. Virol. 67:3172.1993). For use in accordance with this invention, the alphavirus vectorgenome is substituted for the P1 capsid sequences and used as a meansfor providing polio-pseudotyped alphavirus vectors after transfecting invitro transcribed alphavirus vector RNA transcripts into the cell line.Conversely, alphavirus structural proteins also may be substituted forthe VP2 and VP3 proteins, subsequently providing an alternativepackaging cell line system for alphavirus based vectors.

In an alternative system, several components are used, including: (1)alphavirus structural proteins made in the baculovirus system usingtechniques described by Smith et al. (supra) (or in other proteinproduction systems, such as yeast or E. coli); (2) viral vector RNA madein the known T7, SP6 or other in vitro RNA-generating system (Flamant etal., J. Virol. 62:1827, 1988); (3) tRNA transcribed in vitro or purifiedfrom yeast or mammalian tissue culture cells; (4) liposomes (withembedded envelope glycoproteins); and (5) cell extract or purifiednecessary components when identified (typically from mouse cells) toprovide RNA processing, and any or other necessary cell-derivedfunctions.

Within this procedure, components (1), (2) and (3), from above, aremixed, and then envelope glycoprotein associated alphavirus proteins,cell extract and pre-liposome mix (lipid in a suitable solvent) areadded. In an alteration of the procedure, the alphavirus envelopeglycoproteins are embedded in the liposomes prior to addition to themixture of (1), (2), and (3). The resulting mixture is then treated(e.g., by sonication, temperature manipulation, or rotary dialysis) toallow envelopment of the viral nucleocapsid particles with lipid plusembedded alphavirus envelope glycoprotein in a manner similar to thatfor liposome encapsidation of pharmaceuticals (Gould-Fogerite et al.,Anal. Biochem. 148:15, 1985). This or similar procedures can be used toproduce high titres of packaged alphavirus vectors without therequirement of establishing intermediate packaging cell lines.

Example 9 Cell Line or Tissue Specific Alphavirus Vectors—“HybridEnvelopes”

The tissue and cell-type specificity of alphaviruses is determinedprimarily by the virus-encoded envelope proteins, E1 and E2. Thesevirion structural proteins are transmembrane glycoproteins embedded in ahost cell-derived lipid envelope that is obtained when the viralparticle buds from the surface of the infected cell. The envelopesurrounds an icosahedral nucleocapsid, comprised of genomic RNAcomplexed with multiple, highly ordered copies of a single capsidprotein. The E1 and E2 envelope glycoproteins are complexed asheterodimers which have been reported to assemble into trimericstructures, forming the characteristic “spikes” on the virion surface.In addition, the cytoplasmic tails of these proteins interact with thenucleocapsids, initiating the assembly of new viral particles (Virology193:424, 1993). Properties ascribed to the individual glycoproteins ofSindbis virus include receptor binding by glycoprotein E2 (Virology181:694, 1991) and glycoprotein E1-mediated fusion of the virionenvelope and the endosomal membrane, resulting in delivery of thenucleocapsid particle into the cytoplasm (New Aspects ofPositive-Stranded RNA Virus, pp. 166-172, 1990).

The present invention recognizes that by disrupting glycoproteinactivity (in particular, but not limited to that of E2) andco-expressing an intact heterologous glycoprotein, or by creating hybridenvelope gene products (i.e., specifically, an alphavirus envelopeglycoprotein having its natural cytoplasmic domain and membrane-spanningdomain, with its exogenous binding domain replaced by the correspondingdomain(s) from a different envelope glycoprotein, or by replacing the E2and/or E1 glycoproteins with those of other alphaviruses or theirderivatives which differ from that of the vector in their tissuetropism, the host range specificity may be altered without disruptingthe cytoplasmic functions required for virion assembly. Alternatively,by replacing one or more of the alphavirus structural proteins with thestructural protein(s) of another virus and introducing the correspondingviral packaging sequence into the alphavirus vector construct, assemblyof recombinant alphavirus vector constructs into particles of othervirus types can be achieved. Thus, recombinant alphavirus particles canbe produced which have an increased affinity for pre-selected targetcells, depending on the tropism of the protein molecule(s) or domain(s)introduced.

In one embodiment, substitution of the analogous envelop glycoproteinsE1 and/or E2 from other alphaviruses or their variants is performed toalter tissue tropism. For example, Venezuelan equine encephalitis virus(VEE) is an alphavirus which exhibits tropism for cells of lymphoidorigin, unlike its Sindbis virus counterpart. Therefore, Sindbis-derivedvector constructs packaged in a cell line expressing the VEE structuralproteins display the same lymphotropic properties as the parental VEEvirus from which the packaging cell structural protein gene cassette wasobtained.

Specifically, the Trinidad donkey strain of VEE virus (ATCC #VR-69) ispropagated in BHK-21 cells, and virion RNA is extracted using proceduressimilar to those described in Example 1. The entire structural proteincoding region is amplified with a primer pair whose 5′-ends map,respectively, to the authentic AUG translational start site, includingthe surrounding Kozak consensus sequence, and UGA translational stopsite. The forward primer is complementary to VEE nucleotides 7553-7579,and the reverse primer is complementary to VEE nucleotides 11206-11186(sequence from Kinney et al., Virology 170:19-30, 1989). PCRamplification of VEE cDNA corresponding to the structural protein genesis accomplished using a two-step reverse transcriptase-PCR protocol asdescribed above, the VEE genome RNA as template, and the followingoligonucleotide pair:

Forward primer (VEE 7553F):

5′-TATATATATGCGGCCGCACCGCCAAGATGTTCCCGTTCCAGCCA-3′ (SEQ. ID NO. 90)

Reverse primer (VEE 11206R):

5′-TATATATATGCGGCCGCTCAATTATGTTTCTGGTTGGT-3′ (SEQ. ID NO. 91)

In addition to their respective complementarities to the indicated VEEnucleotides, each primer includes a Not I recognition sequence at their5′ ends. Following PCR amplification, the 3800 bp fragment is purifiedin a 1% agarose gel and digested with the enzyme Not I. The resultingfragment is then ligated separately into the pOP13 and pOPRSV1 vectors(Stratagene) described previously, which are digested with Not I andtreated with calf intestinal alkaline phosphatase. These resultingvectors, which contain the entire VEE structural protein codingsequence, are known as pOP13-VEESP and pOPRSV1-VEESP. The use of theseclones in the development of VEE-based packaging cell lines follows thatdescribed for Sindbis packaging lines. Alternatively, the PCR amplifiedVEE structural protein gene fragment digested with NotI is ligated intothe replicon inducible ELVIS cassette described in Example 7. PlasmidpVGELVISBV-linker is digested with Bsp EI to remove most nonstructuralprotein coding sequences, and the vector is then re-ligated with itselfto generate the construct pVGELVISdl-linker. Subsequently, this plasmidis digested with NotI, treated with calf intestinal alkalinephosphatase, and ligated with the NotI digested VEE fragment to generatethe expression cassette pVGELVdlVEE. Plasmid DNA of this construct istransfected into the appropriate cell line and selection for G418resistance is performed as described in Example 7. In addition,variations of the vector-inducible or lac operon-VEE structural proteingene expression vectors may be constructed using other systems describedherein. Additionally, other variations may be constructed which combinethe capsid protein gene of one alphavirus (for example, Sindbis) withthe envelope glycoprotein genes of another alphavirus (for example, VEE)in a split gene approach, as described in Example 7. Furthermore,variants of VEE, and other alphaviruses and their variants differing intissue tropism, are useful when following this approach.

In another embodiment, a RNA packaging signal derived from another virusis inserted into the alphavirus vector to allow packaging by thestructural proteins of that corresponding virus. For example, the 137nt. packaging signal from hepatitis B virus, located between nts. 3134and 88 and spanning the precore/core junction (Junker-Niepmann et al.EMBO J. 9:3389, 1990), is amplified from an HBV template using twooligonucleotide primers. PCR is performed using a standard threetemperature cycling protocol, plasmid pHBV1.1 (Junker-Niepmann et al.EMBO J. 9:3389, 1990) as the template, and the following oligonucleotidepair, each of which contain 20 nucleotides complementary to the HBVsequence and flanking ApaI recognition sequences:

Forward primer (HBVpkgF):

5′-TATATGGGCCCTACATGTCCCACTGTTCAAG-3′ (SEQ. ID NO. 117)

Reverse primer (HBVpkgR):

5′-TATATGGGCCCGTACGGAAGGAAAGAAGTCA-3′ (SEQ. ID NO. 118)

Following amplification, the PCR amplicon is digested with ApaI andpurified from a 1.5% agarose gel using MERMAID™ (Bio101). Sindbis vectorplasmid pKSSINdlJRsjrc (Example 3) also is digested with ApaI, underlimited conditions to cleave at only one of its two sites, followed bytreatment with CIAP, purification from a 1% agarose gel, and ligationwith the above-synthesized HBV amplicon, to produce a constructdesignated pKSSINhbvJR. Other alphavirus vectors (see Example 3) arereadily modified in a similar manner. Cell lines which express the HBVcore, preS/S, and P proteins necessary for packaging of the RNA sequenceare derived by modification of helper plasmid pCH3143 (Junker-Niepmannet al., EMBO J. 9:3389, 1990) to include a selectable marker. Anexpression cassette containing the neomycin resistance marker isobtained by digestion of plasmid pBK-RSV (Stratagene) with Mst II andblunt-ending with Klenow fragment. The selectable marker is then ligatedinto any of several unique sites within pCH3143 that have been digestedand their termini made blunt. The resulting construct is transfectedinto a desired cell line, for example, mouse hepatoma line Hepa1-6 (ATCC#CRL1830), and selected using the drug G418, as described in Example 7.Introduction of the pKSSINhbvJR vector, or related RNA- or DNA-basedalphavirus vectors, results in the production of packaged vectorparticles with the same hepatotropism as HBV.

Similarly, the packaging signal from a coronavirus can be incorporatedinto the alphavirus vector. For example, the 190 nt packaging signalfrom mouse hepatitis virus (MHV), comprising nts 2899 to 3089 (Fosmireet al., J. Virol. 66:3522, 1992), is amplified in a standard three cyclePCR protocol using THERMALASE™ polymerase, DIssF plasmid MP51-2 (Fosmireet al., J. Virol. 66:3522, 1992) as the template, and the followingoligonucleotides, which contain flanking ApaI recognition sites:

Forward primer (MHVpkgF):

5′-TATATGGGCCCATTTTGGTTTTGCTATGCGTA-3′ (SEQ. ID NO. 119)

Reverse primer (MHVpkgF):

5′-TATATGGGCCCATCGAGGTGAGAAAGAGGAC-3′ (SEQ. ID NO. 125)

Following amplification, the PCR amplicon is digested with ApaI,purified from a 1.5% agarose gel using MERMAID™, and ligated intopKSSINdlJRsjrc, prepared as described for HBV. The resulting constructis designated pKSSINmhvJR. Other alphavirus vectors (see Example 3) arereadily modified in a similar manner. Packaging of vectors modified withthis MHV sequence is accomplished by using expression cassettes whichproduce each of the required coronavirus structural proteins:nucleocapsid (N protein; Armstrong et al., NAR 11:883, 1983); membrane(M protein, Armstrong et al., Nature 308:751, 1984); and spike (Sprotein, Luytjes et al., Virology 161:479, 1987). Preferably, theseproteins are inserted into the vector-inducible pVGELVSdl-linker plasmid(described previously in this example) and selected for with the G418drug following transfection into the appropriate cell type. Otherexpression methodologies (see Example 7) may also be readily utilized.Additional coronaviruses, for example, human coronaviruses OC43 (ATCC#VR-759) and 229E (ATCC #VR-740), can be readily used in place of MHV toproduce packaged recombinant alphavirus particles which show tropism forcells in the respiratory tract.

Similarly, the packaging signal from a retrovirus can be incorporatedinto an alphavirus vector construct. For example, the 351 nt extendedpackaging signal (ψ+) from Mo-MLV, corresponding to nts 212 to 563 (Mannet al., Cell 33:153, 1983), is amplified in a standard three cycle PCRprotocol as described above, using plasmid pMLV-K (Miller, J. Virol.49:214, 1984) as template and the following oligonucleotides, each ofwhich contain a flanking ApaI recognition site:

Forward primer (MLVpkgF):

5′-TATATGGGCCCTGTATCTGGCGGACCCGTGG-3′ (SEQ. ID NO. 126)

Reverse primer (MLVpkgR):

5′-TATATGGGCCCGCAGACAAGACGCGCGGCGC-3′ (SEQ. ID NO. 127)

Following amplification, the PCR amplicon is digested with ApaI,purified from a 1.5% agarose gel using GENECLEAN™, and ligated intoplasmid pKSSINdlJRsjrc, prepared as described above. The resultingconstruct is designated pKSSINmlvJR. Other alphavirus vectors (seeExample 3) are readily modified in a similar manner. The generation of aretroviral-derived producer cell line for packaging and production ofthe above alphavirus vector constructs is accomplished by transfectingan appropriate packaging cell line, for example amphotropic line DA (WO92/05266), and selecting for resistance to the drug G418, as describedpreviously.

In each case, the packaging sequences from HBV, coronavirus, retrovirus,or any other virus, also may be incorporated into alphavirus vectors atlocations other than those outlined above, provided the location is notpresent in the subgenomic transcript. For example, the next mostpreferable site of insertion is the carboxy-terminal region ofnonstructural protein 3, which has been shown to be highly variable inboth length and sequence among all alphaviruses for which sequenceinformation is available. Further, these applications are not limited bythe ability to derive the corresponding packaging cell lines, as thenecessary structural proteins also may be expressed using any of thealternative approaches described in Example 8.

In yet another embodiment, a heterologous glycoprotein or cellularligand is expressed in the lipid bilayer of a packaging cell line forproducing enveloped recombinant alphavirus particles. This approach issimilar to that described in Example 8 for the production of VSV-Gpseudo-typed alphavirus vectors, except that in this configuration, theE2 receptor-binding function is inactivated by insertion, deletion, orsite-specific mutagenesis. As an example, receptor binding function ofE2 can be inactivated by techniques known in the art to restrict vectorparticle tropism to that which is supplied by the heterologousglycoprotein or cellular ligand. In addition to the example of VSV-Gpseudo-typing, other viral glycoproteins which target specific cellularreceptors (such as the retroviral HIV gp120 protein for CD4 celltargeting) are utilized when expressed from standard vectors stablytransfected into alphavirus packaging cell lines.

In another configuration, chimeric glycoproteins are prepared whichallow for targeting of alphavirus vector constructs into particular celllines in vitro or tissue types in vivo. To construct such a chimericglycoprotein, specific oligonucleotides encoding the ligand bindingdomain of the desired receptor, plus homologous alphavirus sequences(which include a unique specific restriction endonuclease site), areused to amplify an insert sequence that can be substituted into analphavirus structural protein expression cassette. Alternatively,limited Bal-31 digestions from a convenient restriction enzyme site areperformed in order to digest back to a permissive insertion site,followed by blunt end ligation of a fragment encoding a small receptorbiding domain, an entire viral glycoprotein, or cell surface ligand. Asan example, peptides corresponding to the principal neutralizing domainof the HIV gp120 envelope protein Binding to CD4 of Synthetic PeptidesPatterned on the Principal neutralizing Domain of the HIV-1 EnvelopeProtein, Autiero, et al, Virology 185, 820-828, 1991) can be used todisrupt normal E2 tropism and provide CD4 cell targeting.

While inclusion of the HIV gp120 neutralizing domain illustrates oneexample of a hybrid or chimeric envelope protein, the possibilities arenot limited to viral glycoproteins. For example, the receptor bindingportion of human interleukin-2 can be combined with the envelopeprotein(s) of an alphavirus to target vectors to cells with IL-2receptors. Furthermore, the foregoing technique can be used to create arecombinant alphavirus particles with envelope proteins that recognizeFc portions of antibodies. Monoclonal antibodies which recognize onlypreselected target cells are then bound to such Fc receptor-bearingalphavirus vector particles, such that the vector particles bind to andinfect only those preselected target cells (for example, tumor cells).Alternatively, a hybrid envelope with the binding domain of avidin isused to target cells that have been coated with biotinylated antibodiesor other ligands. The patient is first flooded with antibodies, and thenallowed time to clear unbound and nonspecifically-bound antibody beforeadministering the vector. The high affinity (10⁻¹⁵) of the avidinbinding site for biotin will allow accurate and efficient targeting tothe original tissue identified by the monoclonal “image”. Additionaltargeting approaches are known in the art and can readily be adopted foruse in the practice of the present invention. For example, see U.S. Ser.No. 08/242,407.

Example 10 Lactose Formulation of a Recombinant Alphavirus Vector

Crude recombinant alphavirus particles are obtained from a Celliganbioreactor (New Brunswick, N.J.) containing packaging cells transfectedor transduced with the alphavirus vector construct, and bound to thebeads of the bioreactor matrix. The cells release the packagedrecombinant alphavirus particles into growth media that is passed overthe cells in a continuous flow process. The media exiting the bioreactoris collected and passed initially through a 0.8 micron filter, thenthrough a 0.65 micron filter to clarify the crude recombinant alphavirusparticles. The filtrate is concentrated utilizing a cross flowconcentrating system (Filtron, Boston, Mass.). Approximately 50 units ofDNase (Intergen, New York, N.Y.) per ml of concentrate is added todigest exogenous DNA. The digest is diafiltrated using the same crossflow system to 150 mM NaCl, 25 mM tromethamine, pH 7.2. The diafiltrateis loaded onto a Sephadex S-500 gel column (Pharmacia, Piscataway,N.J.), equilibrated in 50 mM NaCl, 25 mM tromethamine, pH 7.4. Thepurified recombinant alphavirus particles are eluted from the SephadexS-500 gel column in 50 mM NaCl, 25 mM tromethamine, pH 7.4.

The formulation buffer containing lactose is prepared as a 2Xconcentrated stock solution. The formulation buffer contains 25 mMtromethamine, 70 mM NaCl, 2 mg/ml arginine, 10 mg/ml human serum albumin(HSA), and 100 mg/ml lactose in a final volume of 100 mls at a pH 7.4.

The purified recombinant alphavirus particles are formulated by addingone part 2X lactose formulation buffer to one part S-500 purifiedrecombinant alphavirus particle preparation. The formulated recombinantalphavirus particles can be stored at −70° C. to −80° C. or dried.

The formulated alphavirus particles are lyophilized in an EdwardsRefrigerated Chamber (3 Shelf RC3S unit) attached to a Supermodulyo 12Kfreeze dryer (Edwards High Vacuum, Tonawanda, N.Y.). When the freezedrying cycle is completed, the vials are stoppered under a vacuumfollowing a slight nitrogen gas bleeding. Upon removal, vials arecrimped with aluminum seals. The lyophilized recombinant alphavirusparticles are reconstituted with 1.0 ml water or other physiologicallyacceptable diluent.

Example 11 Administration of Recombinant Alphavirus Particles

A therapeutic alphavirus vector used for the treatment of Gaucherdisease (see Example 17) may be administered by transducing autologousCD34⁺ cells in an ex vivo protocol or by direct injection of the vectorinto the patient's bone marrow. In order to achieve the longesttherapeutic expression of GC from the recombinant multivalent vector,the best mode of administration is to transduce long lived cellprecursors of the clinically affected cell type, for example monocytesor macrophages. By transducing the earliest precursors of the effectedcell type, the cell precursors are able to self renew and repopulate theperipheral blood with maturing GC positive cells. The earliestpluripotent hematopoietic stem cell studied to date are the CD34⁺ cellswhich make up 1%-4% of a healthy bone marrow population or 0.1% in theperipheral blood population. Being able to transduce CD34⁺ cells isimportant in sustaining long term expression not only for themonocyte/macrophage lineage but any hematopoietic cell targeted for atherapeutic protein. Two approaches for transducing CD34⁺ cells includean ex vivo and an in vivo protocol. The in vivo protocol focuses ontransducing an indiscriminate population of bone marrow cells by directinjection of the vector into the bone marrow of patients. The ex vivoprotocol focuses on isolating CD34⁺ positive stem cells, from thepatient's bone marrow, or an infant patient's umbilical cord blood,transducing the cells with vector, then subsequently injecting theautologous cells back into the patient. Both approaches are feasible,but the ex vivo protocol enables the vector to be used most efficientlyby transducing a specific cultured population of CD34⁺ cells. Details ofan ex vivo method are provided in the following section.

EX VIVO ADMINISTRATION OF A MULTIVALENT GC SINDBIS VECTOR

CD34⁺ cells are collected from the patient's bone marrow by a syringeevacuation performed by a physician familiar with the technique.Alternatively, CD34⁺ cells may also be obtained from an infant'sumbilical cord blood if the patient is diagnosed before birth.Generally, if the bone marrow is the source of the CD34⁺ cells, 20 bonemarrow aspirations are obtained by puncturing femoral shafts or from theposterior iliac crest under local or general anesthesia. Bone marrowaspirations are then pooled, suspended in Hepes-Buffered Hanks' balancedsalt solution containing heparin at 100 units per ml anddeoxyribonuclease I at 100 ug/ml and then subjected to a Ficoll gradientseparation. The buffy coated marrow cells are then collected and washedaccording to CellPro's CEPRATE® LC (CellPro, Bothell, Wash.) (CD34)Separation system (see U.S. Pat. Nos. 5,215,927; 5,225,353; 5,262,334;5,215,926 and PCT/US91/07646). The washed buffy coated cells are thenstained sequentially with anti-CD34 monoclonal antibody, washed thenstained with biotinylated secondary antibody supplied with CEPRATE®system. The cell mixture is then loaded onto the CEPRATE® avidin column.The biotin-labeled cells are adsorbed onto the column while unlabeledcells passed through. The column is then rinsed according to theCEPRATE® system directions and CD34⁺ cells eluted by agitation of thecolumn by manually squeezing the gel bed. Once the CD34⁺ cells arepurified, the purified stem cells are counted and plated at aconcentration of 1×10⁵ cells/ml in Iscove's modified Dulbecco's medium(IMDM; Irvine Scientific, Santa Ana, Calif.) containing 20% poolednon-heat inactivated human AB serum (hAB serum).

After purification, several methods of transducing purified stem cellsmay be performed. One approach involves immediate transduction of thepurified stem cell population with recombinant alphavirus particlescontained in culture supernatants derived from vector packaging orproducing cells. A second approach involves co-cultivation of anirradiated monolayer of vector producing cells with the purifiedpopulation of nonadherent CD34⁺ cells. A third approach involves asimilar co-cultivation approach, however, the purified CD34⁺ cells areprestimulated with various cytokines and cultured 48 hours prior to theco-cultivation with the irradiated vector producing cells. Sincealphavirus vectors are able to infect nonreplicating cells,prestimulation of these cells may not be required, howeverprestimulation of these cultures causing proliferation will provideincreased cell populations for reinfusion into the patient.

Prestimulation of the CD34⁺ cells is performed by incubating the cellswith a combination of cytokines and growth factors which include IL-1,IL-3, IL-6 and mast cell growth factor (MGF). Prestimulation isperformed by culturing 1-2×10⁵ CD34⁺ cells/ml of medium in T25 tissueculture flasks containing bone marrow stimulation medium for 48 hours.The bone marrow stimulation medium consists of IMDM containing 30%non-heat inactivated hAB serum, 2 mM L-glutamine, 0.1 mM2-mercaptoethanol, 1 M hydrocortisone, and 1% deionized bovine serumalbumin. All reagents used in the bone marrow cultures should bescreened for their ability to support maximal numbers of granulocyte,erythrocyte, macrophage, megakaryoctye, colony-forming units from normalmarrow. Purified recombinant human cytokines and growth factors (ImmunexCorp., Seattle, Wash.) for prestimulation should be used at thefollowing concentrations: E. coli-derived IL-1 (100 U/ml), yeast-derivedIL-3 (5 ng/ml), IL-6 (50 U/ml), and MGF (50 ng/ml) (Anderson et al.,Cell Growth Differ. 2:373, 1991).

After prestimulation of the CD34⁺ cells, they are then infected byco-cultivation with the irradiated Sindbis producer cell line(expressing the GC therapeutic vector) in the continued presence of thestimulation medium. The Sindbis vector producing cell line is firsttrypsinized, irradiated (10,000 Rads) and replated at 1-2×10⁵ cells/mlof bone marrow stimulation medium. The following day, 1-2×10⁵prestimulated CD34+ cells/ml is added to the Sindbis vector producingcell line monolayer. Co-cultivation of the cells is performed for 48hours. After co-cultivation, the CD34⁺ cells are collected from theadherent Sindbis vector producing cell monolayer by vigorous washingwith medium and plated for 2 hours to allow adherence of any dislodgedvector producing cells. The CD34+ cells are collected and expanded foran additional 72 hours. The cells are then harvested and frozen inliquid nitrogen using a cryo-protectant in aliquots of 1×10⁷ cells pervial. Once the treated CD34⁺ cells have been tested for the absence ofadventitious agents, frozen transformed CD34⁺ cells may be thawed,plated to a concentration of 1×10⁵ cells/ml and cultured for anadditional 48 hours in bone marrow stimulation medium. Transformed cellsare collected, washed twice and resuspended in normal saline. The numberof transduced cells used to infuse back into the patient per infusion isprojected to be at a minimum of 1-10×10⁷ cells per patient per injectionsite requiring up to four injection sites. Infusion may be performeddirectly back into the patient's bone marrow or directly into theperipheral blood stream. Patients receiving autologous transduced bonemarrow cells may be either partially or whole body irradiated, todeplete existing bone marrow populations. Treatment may be assessed atvarious time points post infusion to determine GC activity and forlength of expression in differentiated cell types. If at some pointduring the course of follow-up procedures expression decreases or isnonexistent, transduced autologous cells may be reinjected into thepatient.

Example 12 Determination of Vector Units in a Preparation by Infectionof a Reporter Protein Expressing Cell Line Under the Control of theSindbis Junction Region Determination of Vector Units in a Preparationby Infection of a β-Galactosidase Expressing Reporter Cell Line

In order to administer the proper therapeutic dose of vector toindividuals, it is desirable to derive a method by which the vectorinfectious units contained in a preparation can be determined easily.This is accomplished by the generation of a cell line which expressesβ-galactosidase or another reporter gene only when functional Sindbisnonstructural proteins are present in the cell. The cell line can beinfected with increasing dilutions of a Sindbis vector preparation suchthat individual cells are not infected with more than one vectorparticle, allowing the titer, or vector units, to be determined. Thus,the cell line is an assay of function particles present in a vectorpreparation.

A. GENERATION OF A CELL LINE WHICH EXPRESSES FUNCTION β-GALACTOSIDASEPROTEIN UNDER THE CONTROL OF SINDBIS NONSTRUCTURAL PROTEINS

In one configuration, a eukaryotic expression cassette is constructedwhich contains a 5′-end sequence capable of initiating transcription ofSindbis RNA, a Sindbis junction region, a reporter gene, and a 3′-endSindbis RNA polymerase recognition sequence for minus-strand synthesis.This cassette is positioned in an antisense orientation, adjacent to aeukaryotic transcriptional promoter. Additionally, these constructs alsomay contain a catalytic ribozyme sequence immediately adjacent toSindbis nucleotide 1 of the 5′-end sequence which will result incleavage of the primary RNA transcript precisely after this Sindbisnucleotide. In this antisense orientation, the reporter gene cannot betranslated and is dependent entirely on the presence of Sindbisnonstructural proteins for transcription into positive stranded mRNAprior to reporter gene expression. These non-structural proteins will beprovided by the Sindbis vector preparation being titered. In addition,this configuration, if designed to contain the precise Sindbis genome5′- and 3′-end sequences, will allow for the reporter gene transcriptsto undergo amplification by utilizing the same nonstructural proteinsprovided by the Sindbis vector.

An example of this antisense titering construction is as follows.Briefly, the plasmid pKSSINBV-lacZ (described in Example 6) is digestedwith the enzymes Apa I and Bam HI. This results in the removal of theSindbis 5′ and Sindbis nonstructural protein sequences. The 7 kbpfragment is purified on a 0.7% agarose gel. This fragment is ligated toa fragment obtained by digestion of pd5′26s (described in Example 7)with ApaI and BamHI followed by gel purification of the 0.4 kbp fragmentcontaining the HDV ribozyme and 5′ Sindbis sequences. The resultingconstruct is known as pKSd5′BV-lacZ. pKSd5′BV-lacZ is digested with ApaI and Pme I followed by purification of the 7.4 kbp fragment on a 0.7%agarose gel. This fragment contains the HDV ribozyme, Sindbis 5′ end,junction region, LacZ gene, and Sindbis 3′ end sequences. This fragmentis ligated in the antisense orientation into pcDNA3 (Promega Corp.,Madison, Wis.) by digestion of pcDNA3 with Apa I and EcoRV followed byGENECLEAN™ purification. The resulting construct, containing a CMVpromoter which transcribes an antisense reporter cassette RNA of theconfiguration Sindbis 3′-end sequence/LacZ gene/junction region/Sindbis5′-end sequence/HDV ribozyme, is known as pSINjra-gal.

BHKSINjra-gal cells are derived by transfection of 5×10⁵ BHK-21 cells,grown in a 60 mm petri dish, with 5 ug of the pSINjra-gal vectorcomplexed with the polycation reagent Transfectam™ (Promega, Madison,Wis.). At 24 hour post-transfection, the media is supplemented with 400ug/ml of G418 (GibcoBRL, Gaithersburg, Md.). After all non-transfectedcells have died and G418 resistant colonies have begun dividing, thecells are removed from the plate by trypsinization, pooled, then clonedby limiting dilution. Several clones are tested for the production offunctional β-galactosidase by infection with a known titer of awild-type stock of Sindbis virus. Production of functionalβ-galactosidase in candidate BHKSINjra-gal clones is determined 6 hourspost-infection by first fixing PBS-rinsed cells with a solutioncontaining 2% formaldehyde (37% stock solution)/0.2% glutaraldehyde,then staining the cells with a solution containing 0.5 mM potassiumferricyanide/0.5 mM potassium ferrocyanide/2 mM MgCl₂/1 mg/ml X.gal.Blue cells are clearly visible within 3 hours. Provided that the Sindbisvirus stock does not contain a high level of defective interfering (DI)particles, the virus titer as determined by plaque assay on BHK-21 cellsshould be similar to the titer observed by X-gal staining onBHKSINjra-gal cells.

The titer of various alphavirus vector preparations, in vector units,produced from packaging cell lines such as those described in Example 7,is determined by infection of confluent monolayers of BHKSINjra-galcells with several dilutions of vector. The titer of the vectorpreparation is determined at 6 hour post-infection by visualization ofcells producing β-galactosidase protein, as described above. Since thealphavirus vectors described do not contain the viral regioncorresponding to the structural genes, it is not possible to determinethe titer of a vector preparation by plaque assay in BHK-21 cells.

Alternatively, a titering cell line is produced by using a differentreporter cassette configuration, which consists of a eukaryoticpromoter/5′-end Sindbis sequence recognized by the viraltranscriptase/Sindbis junction region/reporter gene/Sindbis RNApolymerase recognition sequence for minus-strand synthesis, and isexpressed in a sense-orientation. This reporter expression cassetterequires synthesis, by vector-supplied Sindbis nonstructural proteins,into an antisense RNA molecule, prior to transcription of the subgenomicmessage encoding the reporter gene.

Specifically, the sense-orientation packaging construct is created asfollows. Plasmid pVGELVIS is digested with the enzyme Apa I, whichcleaves at nucleotide 11737, just downstream of the Sindbis 3′-end. TheApa I-digested DNA is blunt-ended by the addition of T4 DNA polymeraseand dNTPs and incubation at 16° C. for 10 minutes. After heatinactivation of the polymerase, the DNA fragment is digested with theenzyme Sfi I, and the 10041 bp fragment is purified in a 1% agarose gel.Plasmid pSKSINBV-lacZ is digested with the enzymes Pme I and Sfi I. The6.4 kbp fragment is purified in a 1% agarose gel. The 6.4 kbppSKSINBV-lacZ fragment then is ligated into the purified pVGELVISfragment to create the plasmid pELVIS-gal. This plasmid contains thecomplete Sindbis nonstructural proteins, Sindbis junction region, LacZgene and Sindbis 3′-end replicase recognition sequence under the controlof the MuLV LTR promoter. Plasmid pELVIS-gal is digested with Bsp EI,purified by GENECLEAN (Bio 101 Corp., San Diego, Calif.) and religatedto itself. Bsp EI removes the Sindbis nonstructural protein genesequences between nts 422-7054. The re-ligated construct contains a 5′sequence that is capable of initiating transcription of Sindbis RNA,Sindbis junction region, sequences encoding the LacZ gene, the Sindbis3′-end sequences required for synthesis of the minus-strand RNA, alldownstream, and under the transcriptional control of a MuLV-LTRpromoter. This construct is known as pELVISdlNSP-gal.

Plasmid pELVISdlNSP-gal is transfected into BHK-21 cells and tested asdescribed previously. The BHK pELVISdlNSP-gal cells produces an RNAtranscript with a 5′-end sequence that is recognized by the Sindbistranscriptase, a Sindbis junction region, sequences encoding the LacZgene, and Sindbis 3′-end sequences required for synthesis of theminus-strand RNA. β-galactosidase expression from the primary transcriptis prevented because of an upstream open-reading frame and stop codonscreated by the Bsp EI deletion. The addition of Sindbis nonstructuralproteins, provided by the Sindbis vector being titered, results intranscription of active LacZ transcripts from the Sindbis junctionregion, after initial synthesis of an antisense intermediate.Furthermore, this configuration, if designed to contain the preciseSindbis genome 5′- and 3′-end sequences, allows the reporter genetranscripts to undergo amplification by utilizing the same nonstructuralproteins provided by the Sindbis vector.

In another configuration, a titering cell line is produced using anexpression cassette containing an antisense reporter gene followed bythe 3′-end alphavirus replicase recognition sequences, positioned in thesense-orientation. This construct, under the control of a eukaryoticpromoter, produces an RNA transcript that is recognized and transcribedby alphavirus nonstructural proteins provided by the vector to betitered. The alphavirus nonstructural proteins recognize sequences inthe primary reporter transcript, and in turn, synthesize a sensereporter transcript. This construct does not benefit from amplificationof the reporter gene transcript, but should still provide sufficienttranscripts to allow for vector titering.

Construction of this type of titering cassette is as follows. Briefly,pSV-β-galactosidase vector (Promega Corp., Madison, Wis.) is digestedwith the enzyme Hind III and blunt-ended as described above. The plasmidis further digested with the enzymes Bam HI and Xmn I to remove the LacZgene, and reduce the size of the remaining fragment. The 3737 ntfragment, containing the LacZ gene, is purified in a 1% agarose gel andligated into pcDNA3 (Invitrogen, San Diego, Calif.) that has beendigested with the enzymes Bam HI and Eco RV. The new plasmid constructis known as pcDNAaLacZ. This plasmid is digested with the enzyme Apa I,blunt-ended as above, and further digested with the enzyme Xho I.Plasmid pSKSINBV (described previously) is digested with Sac I,blunt-ended as before, and then digested with Xho I. The resulting 146nt fragment containing the Sindbis 3′ replicase recognition sequence ispurified in a 1.2% agarose gel, ligated into the digested pcDNAaLacZvector. The re-ligated construct contains an antisense LacZ gene and a3′ Sindbis replicase protein recognition sequence downstream from a CMVpromoter. The resulting construct is known as pcDNAaLacZ-3′Sin. Theconstruct is transfected into BHK cells and utilized as describedpreviously.

B. GENERATION OF A CELL LINE WHICH EXPRESSES FUNCTIONAL LUCIFERASEPROTEIN UNDER THE CONTROL OF SINDBIS NONSTRUCTURAL PROTEINS.

An alternate reporter for a titering construct based upon the senseconfiguration of the reporter gene and requiring the nonstructuralproteins for expression utility is luciferase. Again, the non-structuralproteins are supplied in trans by the Sindbis vector preparation beingtitered. To generate this construct, pELVIS-luc is digested with Eco 47III and Hpa I. These digests remove nucleotides 1407-6920 from withinthe non-structural coding region. After heat inactivation of theenzymes, the digested vector is religated under dilute conditions. Thisconstruct is known as pELVISdlE-Hluc. The construct is transfected intoBHK cells and utilized as described previously.

Example 13 Generation of Vector Constructs Which Express HBV Antigensfor the Induction of an Immune Response

A. ISOLATION OF HBV E/CORE SEQUENCE

A 1.8 Kb fragment containing the entire precore/core coding region ofhepatitis B is obtained from plasmid pAM6 (ATCC No 45020) following BamHI digestion and gel purification, and ligated into the Bam HI site ofKS II+ (Stratagene, La Jolla, Calif.). This plasmid is designated KS II+HBpc/c. Xho I linkers are added to the Stu I site of precore/core in KSII+ HBpc/c (at nucleotide sequence 1,704), followed by cleavage withHinc II (at nucleotide sequence 2,592). The resulting 877 base pair XhoI-Hinc II precore/core fragment is cloned into the Xho I/Hinc II site ofSK II+. This plasmid is designated SK+HBe.

B. PREPARATION OF SEQUENCES UTILIZING PCR

1. SITE-DIRECTED MUTAGENESIS OF HBV E/CORE SEQUENCE UTILIZING PCR

The precore/core gene in plasmid KS II+ HB pc/c is sequenced todetermine if the precore/core coding region is correct. This sequencewas found to have a single base-pair deletion which causes a frame shiftat codon 79 that results in two consecutive in-frame TAG stop codons atcodons 84 and 85. This deletion is corrected by PCR overlap extension(Ho et al., Gene 77:51, 1989) of the precore/core coding region inplasmid SK+HBe. Four oligonucleotide primers are used for the 3 PCRreactions performed to correct the deletion.

The first reaction utilizes two primers. The sense primer sequencecorresponds to the nucleotide sequence 1,805 to 1,827 of the adw strainand contains two Xho I restriction sites at the 5′ end. The nucleotidesequence numbering is obtained from Genbank (Intelligenics, Inc.,Mountain View, Calif.).

5′ CTC GAG CTC GAG GCA CCA GCA CCA TGC AAC TTT TT-3′  (SEQ. ID NO. 92)

The second primer sequence corresponds to the anti-sense nucleotidesequence 2,158 to 2,130 of the adw strain of hepatitis B virus, andincludes codons 79, 84 and 85.

5′-CTA CTA GAT CCC TAG ATG CTG GAT CTT CC-3′  (SEQ. ID NO. 93)

The second reaction also utilizes two primers. The sense primercorresponds to nucleotide sequence 2,130 to 2,158 of the adw strain, andincludes codons 79, 84 and 85.

5′-GGA AGA TCC AGC ATC TAG GGA TCT AGT AG-3′  (SEQ. ID NO. 94)

The second primer corresponds to the anti-sense nucleotide sequence fromSK+ plasmid polylinker and contains a Cla I site 135 bp downstream ofthe stop codon of the HBV precore/core coding region.

5′-GGG CGA TAT CAA GCT TAT CGA TAC CG-3′  (SEQ. ID NO. 95)

The third reaction also utilizes two primers. The sense primercorresponds to nucleotide sequence 5 to 27 of the adw strain, andcontains two Xho I restriction sites at the 5′ end.

5′-CTC GAG CTC GAG GCA CCA GCA CCA TGC AAC TTT TT   (SEQ. ID NO. 92)

The second primer sequence corresponds to the anti-sense nucleotidesequence from the SK+ plasmid polylinker and contains a Cla I site 135bp downstream of the stop codon of the HBV precore/core coding region.

5′-GGG CGA TAT CAA GCT TAT CGA TAC CG-3′  (SEQ. ID NO. 96)

The first PCR reaction corrects the deletion in the antisense strand andthe second reaction corrects the deletion in the sense strands. PCRreactions one and two correct the mutation from CC to CCA which occursin codon 79 and a base pair substitution from TCA to TCT in codon 81.Primer 1 contains two consecutive Xho I sites 10 bp upstream of the ATGcodon of HBV e coding region and primer 4 contains a Cla I site 135 bpdownstream of the stop codon of HBV precore/core coding region. Theproducts of the first and second PCR reactions are extended in a thirdPCR reaction to generate one complete HBV precore/core coding regionwith the correct sequence.

The PCR reactions are performed using the following cycling conditions:The sample is initially heated to 94° C. for 2 minutes. This step,called the melting step, separates the double-stranded DNA into singlestrands for synthesis. The sample is then heated at 56° C. for 30seconds. This step, called the annealing step, permits the primers toanneal to the single stranded DNA produced in the first step. The sampleis then heated at 72° C. for 30 seconds. This step, called the extensionstep, synthesizes the complementary strand of the single stranded DNAproduced in the first step. A second melting step is performed at 94° C.for 30 seconds, followed by an annealing step at 56° C. for 30 secondswhich is followed by an extension step at 72° C. for 30 seconds. Thisprocedure is then repeated for 35 cycles resulting in the amplificationof the desired DNA product.

The PCR reaction product is purified by 1.5% agarose gel electrophoresisand transferred onto NA 45 paper (Schleicher and Schuell, Keene, N.H.).The desired 787 bp DNA fragment is eluted from the NA 45 paper byincubating for 30 minutes at 65° C. in 400 l high salt buffer (1.5 MNaCl, 20 mM Tris, pH 8.0, and 0.1 mM EDTA). Following elution, 500 μl ofphenol:chloroform:isoamyl alcohol (25:24:1) is added to the solution.The mixture is vortexed and then centrifuged 14,000 rpm for 5 minutes ina Brinkmann Eppendorf centrifuge (5415L). The aqueous phase, containingthe desired DNA fragment, is transferred to a fresh 1.5 ml microfugetube and 1.0 ml of 100% EtOH is added. This solution is incubated on dryice for 5 minutes, and then centrifuged for 20 minutes at 10,000 rpm.The supernatant is decanted, and the pellet is rinsed with 500 l of 70%EtOH. The pellet is dried by centrifugation at 10,000 rpm under vacuum,in a Savant Speed-Vac concentrator, and then resuspended in 10 ldeionized H₂O. One microliter of the PCR product is analyzed by 1.5%agarose gel electrophoresis. The 787 Xho I-Cla I precore/core PCRamplified fragment is cloned into the Xho I-Cla I site of SK+ plasmid.This plasmid is designated SK+HBe-c. E. coli (DH5 alpha, BethesdaResearch Labs, Gaithersburg, Md.) is transformed with the SK+HBe-cplasmid and propagated to generate plasmid DNA. The plasmid is thenisolated and purified, essentially as described by Birnboim et al. (Nuc.Acid Res. 7:1513, 1979; see also Molecular Cloning: A Laboratory Manual,Sambrook et al. (eds.), Cold Spring Harbor Press, 1989). The SK+HBe-cplasmid is analyzed to confirm the sequence of the precore/core gene(FIG. 4).

2. ISOLATION OF HBV CORE SEQUENCE

The single base pair deletion in plasmid SK+ HBe is corrected by PCRoverlap extension as described above in Example 13B. Briefly, fouroligonucleotide primers are used for the PCR reactions performed tocorrect the mutation.

The first reaction utilizes two primers. The sense primer corresponds tothe nucleotide sequence for the T-7 promoter of SK+HBe plasmid.

5′-AAT ACG ACT CAC TAT AGG G-3′  (SEQ. ID NO. 97)

The second primer corresponds to the anti-sense sequence 2,158 to 2,130of the adw strain, and includes codons 79, 84 and 85.

5′-CTA CTA GAT CCC TAG ATG CTG GAT CTT CC-3′  (SEQ. ID NO. 98)

The second reaction utilizes two primers. The anti-sense primercorresponds to the nucleotide sequence for the T-3 promoter present inSK+HBe plasmid.

5′-3′: ATT AAC CCT CAC TAA AG   (SEQ. ID NO. 99)

The second primer corresponds to the sense nucleotide sequence 2,130 to2,158 of the adw strain, and includes codons 79, 84 and 85.

5′-GGA AGA TCC AGC ATC TAG GGA TCT AGT AG-3′  (SEQ. ID NO. 100)

The third reaction utilizes two primers. The anti-sense primercorresponds to the nucleotide sequence for the T-3 promoter present inSK+HBe plasmid.

5′-ATT AAC CCT CAC TAA AG-3′  (SEQ. ID NO. 101)

The second primer corresponds to the sense sequence of the T-7 promoterpresent in the SK+HBe plasmid.

5′-AAT ACG ACT CAC TAT AGG G-3′  (SEQ. ID NO. 102)

The PCR product from the third reaction yields the correct sequence forHBV precore/core coding region.

To isolate HBV core coding region, a primer is designed to introduce theXho I restriction site upstream of the ATG start codon of the corecoding region, and eliminate the 29 amino acid leader sequence of theHBV precore coding region. In a fourth reaction, the HBV core codingregion is produced using the PCR product from the third reaction and thefollowing two primers.

The sense primer corresponds to the nucleotide sequence 1,885 to 1,905of the adw strain and contains two Xho I sites at the 5′ end.

5′-CCT CGA GCT CGA GCT TGG GTG GCT TTG GGG CAT G-3′  (SEQ. ID NO. 103)

The second primer corresponds to the anti-sense nucleotide sequence forthe T-3 promoter present in the SK⁺ HBe plasmid. The approximately 600bp PCR product from the fourth PCR reaction contains the HBV core codingregion and novel Xho I restriction sites at the 5′ end and Cla Irestriction sites at the 3′ end that was present in the multicloningsite of the SK⁺ HBe plasmid.

5′-ATT ACC CCT CAC TAA AG-3′  (SEQ. ID NO. 104)

Following the fourth PCR reaction, the solution is transferred into afresh 1.5 ml microfuge tube. Fifty microliters of 3 M sodium acetate isadded to this solution followed by 500 μl of chloroform:isoamyl alcohol(24:1). The mixture is vortexed and then centrifuged at 14,000 rpm for 5minutes. The aqueous phase is transferred to a fresh microfuge tube and1.0 ml 100% EtOH is added. This solution is incubated at −20° C. for 4.5hours, and then centrifuged at 10,000 rpm for 20 minutes. Thesupernatant is decanted, and the pellet rinsed with 500 μl of 70% EtOH.The pellet is dried by centrifugation at 10,000 rpm under vacuum andthen resuspended in 10 μl deionized H₂O. One microliter of the PCRproduct is analyzed by 1.5% agarose gel electrophoresis. Theapproximately 600 bp Xho I-Cla I HBV core PCR fragment is cloned intothe Xho I-Cla I site of SK⁺ plasmid. This plasmid is designated SK+HBc.

3. ISOLATION OF HBV X ANTIGEN

A 642 bp Nco I-Taq I fragment containing the hepatitis B virus X openreading frame is obtained from the pAM6 plasmid (adw) (ATCC 45020),blunted by Klenow fragment, and ligated into the Hinc II site of SK⁺(Stratagene, La Jolla, Calif.). E. coli (DH5, Bethesda ResearchLaboratories, Gaithersburg, Md.) is transformed with the ligationreaction and propagated.

Since this fragment can be inserted in either orientation, clones areselected that have the sense orientation with respect to the Xho I andCla I sites in the SK⁺ multicloning site. More specifically, miniprepDNAs are digested with the diagnostic restriction enzyme, Bam HI.Inserts in the correct orientation yield two fragments of 3.0 Kb and 0.6Kb in size. Inserts in the incorrect orientation yield two fragments of3.6 Kb and 0.74 Kb. A clone in the correct orientation is selected anddesignated SK-X Ag.

4. CONSTRUCTION OF SINDBIS VECTORS EXPRESSING HBVE, HBV CORE AND HBV X

Construction of a Sindbis vector expressing the HBVe sequence isaccomplished by digesting the SK+HB e-c plasmid with Xho I and Xba I torelease the cDNA fragment encoding HBVe-c sequences. The fragment isthen isolated by agarose gel electrophoresis, purified by GENECLEAN™,and inserted into pKSSINBV (see Example 3), prepared by digestion withXho I and Xba I, and treated with CIAP. This vector is designatedpKSSIN-HBe. Similar vectors may also be made from other Sindbis vectorsdescribed in Example 3, such as, for example, pKSSINd1JRsjrc,pKSSINd1JRsjrPC, pKSSINd1JRsjrNP(7582-7601) and pKSSINd1JRsexjr.

Construction of a Sindbis vector expressing the HBV core sequence isaccomplished by digestion of plasmid SK+HBc (described above) with Xho Iand Xba I. The HBc fragment is isolated by agarose gel electrophoresis,purified by GENECLEAN™ and ligated into pKSSINBV at the Xho I and Xba Isites. This Sindbis-HBc vector is designated pKSSIN-HBc.

Construction of a Sindbis vector expressing the HBV-X antigen sequenceis accomplished by digesting the plasmid SK-X Ag with Xho I and Xba I torelease a cDNA fragment encoding HBV-X sequences. The fragment isisolated by agarose gel electrophoresis, purified using GENECLEAN™, andinserted into pKSSINBV, pre-treated with Xho I and Xba I enzymes. ThisSindbis-HBx vector is designated pKSIN-HBx.

The above Sindbis HBV expressing vectors may also be modified tocoexpress a selectable drug resistance marker dependent on therequirements of the experiment or treatment of the vector infectedcells. In particular, any of the above Sindbis HBV expression vectorsdescribed may also be designed to coexpress G418 resistance. This isaccomplished by incorporating an internal ribosomal entry site (Example5) followed by the bacterial noemycin phosphotransferase gene placed 3′of the HBV coding sequences and 5′ of the terminal 3′ end of the vectorusing the multiple cloning site of the vector. These G418 resistantvector constructs can be used for selecting vector infected cells forthe generation of HBV specific CTL targets in the following sections.

D. EXPRESSION IN INFECTED CELLS WITH SINDBIS VECTORS

1. ELISA

Cell lysates from cells infected by any of the HBV expressing vectorsare made by washing 1.0×10⁷ cultured cells with PBS, resuspending thecells to a total volume of 600 μl in PBS, and sonicating for two5-second periods at a setting of 30 in a Branson sonicator, Model 350(Fisher, Pittsburgh, Pa.) or by freeze thawing three times. Lysates areclarified by centrifugation at 10,000 rpm for 5 minutes.

Core antigen and precore antigen in cell lysates and secreted e antigenin culture supernatant are assayed using the Abbott HBe, rDNA EIA kit(Abbott Laboratories Diagnostic Division, Chicago, Ill.). Anothersensitive EIA assay for precore antigen in cell lysates and secreted eantigen in culture supernatant is performed using the Incstar ETI-EB kit(Incstar Corporation, Stillwater, Minn.). A standard curve is generatedfrom dilutions of recombinant hepatitis B core and e antigen obtainedfrom Biogen (Geneva, Switzerland).

As shown in FIG. 16, using these procedures approximately 100-200 ng/mlHBV e antigen is expressed in the cell lysates and 300-400 ng/ml HBV eantigen is secreted from BHK cells infected with the Sin BV HB e vector.

As shown in FIG. 17, using these procedures, approximately 40 ng/ml HBVcore antigen is expressed in the cell lysates from 10⁶ BHK cellsinfected with the Sin BV HBcore. Mouse fibroblast cells infected withthe recombinant HBcore Sindbis vector express 6-7 fold higher HBV coreprotein levels than the recombinant HBcore retroviral vector transducedcells (WO 93/15207). As shown in FIG. 18, using these procedures,approximately 12-14 ng/ml HBV core antigen is expressed in the celllysates from 10⁶ L-M(TK−) cells infected with the SinBVHBcore vector ascompared to the approximately 2 ng/ml HBV core antigen expressed fromrecombinant HBcore retroviral vector transducer cells.

2. IMMUNOPRECIPITATION/WESTERN BLOT

Characterization of the precore/core and e antigens expressed by vectorinfected cells is performed by immunoprecipitation followed by Westernblot analysis. Specifically, 0.5-1.0 ml of cell lysate in PBS or culturesupernatant is mixed with polyclonal rabbit anti-hepatitis B coreantigen (DAKO Corporation, Carpinteria, Calif.) bound to proteinG-Sepharose (Pharmacia LKB, Uppsala, Sweden) and incubated overnight at4° C. Samples are washed twice in 20 mM Tris-HCl, pH 8.0, 100 mM NaCl,10 mM EDTA and boiled in sample loading buffer with 0.5%2-mercaptoethanol. Proteins are first resolved by SDS polyacrylamide gelelectrophoresis, and then transferred to Immobilon (Millipore Corp.,Bedford, Me.) and probed with the DAKO polyclonal rabbit anti-hepatitisB core antigen, followed by ¹²⁵I-protein A.

E. TESTING IMMUNE RESPONSE

1. CYTOTOXICITY ASSAYS

(a) Inbred Mice

Six- to eight-week-old female C3H/He mice (Charles River, Mass.) areinjected twice intraperitoneally (i.p.) at 1 week intervals with 1×10⁶of Sindbis HBe or HBCore vector. Animals are sacrificed 7 or 14 dayslater and the splenocytes (3×10⁶/ml) cultured in vitro with theirrespective irradiated (10,000 rads) retroviral vector transduced cells(6×10⁴/ml) (WO 93/15207) in T-25 flasks (Corning, Corning, N.Y.).Culture medium consists of RPMI 1640, 5% heat-inactivated fetal bovineserum, 1 mM sodium pyruvate, 50 ug/ml gentamycin and 10⁻⁵M2-mercaptoethanol (Sigma, St. Louis, Mo.). Effector cells are harvested4-7 days later and tested using various effector: target cell ratios in96 well microtiter plates (Corning, Corning, N.Y.) in a standardchromium release assay. Targets are the retroviral vector transducedL-M(TK⁻) cells (ATCC No. CCL 1.3) whereas the non-transduced syngeneiccell lines are used as negative controls. CTL targets may also begenerated by infecting syngeneic cells with the Sindbis HBe or HBcorevector coexpressing the G418 resistance marker. Infected cells are thenselected using 800 g/ml G418 for two weeks. Specifically, Na₂⁵¹CrO₄-labeled (Amersham, Arlington Heights, Ill.)(100 uCi, 1 hour at37° C.) target cells (1×10⁴ cells/well) are mixed with effector cells atvarious effector to target cell ratios in a final volume of 200 μl.Following incubation, 100 ul of culture medium is removed and analyzedin a Beckman gamma spectrometer (Beckman, Dallas, Tex.). Spontaneousrelease (SR) is determined as CPM from targets plus medium and maximumrelease (MR) is determined as CPM from targets plus 1M HCl. Percenttarget cell lysis is calculated as: [(Effector cell+target CPM)−(SR)/(MR) −(SR) ]×100. Spontaneous release values of targets aretypically 10%-20% of the MR.

For certain CTL assays, the effectors may be in vitro stimulatedmultiple times, for example, on day 8-12 after the primary in vitrostimulation. More specifically, 10⁷ effector cells are mixed with 6×10⁵irradiated (10,000 rads) stimulator cells, and 2×10⁷ irradiated (3,000rads) “filler” cells (prepared as described below) in 10 ml of“complete” RPMI medium. (RPMI containing: 5% heat inactivated FetalBovine Serum. two mM L-glutamine, 1 mM sodium pyruvate, 1X non essentialamino acids, and 5×10⁻⁵ M 2-mercaptoethanol). Stimulator cells for invitro stimulation of effector cells are generated from irradiatedretroviral vector transduced (10,000 rads) L-M (TK−) cells. “Filler”cells are prepared from naive syngeneic mouse spleen cells resuspendedin RPMI, irradiated with 3,000 rads at room temperature. Splenocytes arewashed with RPMI, centrifuged at 3,000 rpm for 5 minutes at roomtemperature, and the pellet is resuspended in RPMI. The resuspendedcells are treated with 1.0 ml tris-ammonium chloride (100 ml of 0.17 Mtris base, pH 7.65, plus 900 ml of 0.155 M NH₄Cl; final solution isadjusted to a pH of 7.2) at 37° C. for 3-5 minutes. The secondary invitro restimulation is then cultured for 5-7 days before testing in aCTL assay. Any subsequent restimulations are cultured as described abovewith the addition of 2-10 U of recombinant human IL-2 (200 U/ml, catalog#799068, Boehringer Mannheim, W. Germany).

Using these procedures, it can be shown that CTLs to HBV e antigen canbe induced.

(b) HLA A2.1 Transgenic Mice

Six- to eight-week-old female HLA A2.1 transgenic mice (V. Engelhard,Charlottesville, Va.) are injected twice intraperitoneally (i.p.) at oneweek intervals with 1.0×10⁶ pfu of Sindbis vector expressing HBe orHBcore. Animals are sacrificed 7 days later and the splenocytes(3×10⁶/ml) cultured in vitro with irradiated (10,000 rads) retroviralvector transduced Jurkat A2/K^(b) cells (WO 93/15207), or with peptidecoated Jurkat A2/K^(b) cells (6×10⁴/ml) in flasks (T-25, Corning,Corning, N.Y.). The remainder of the chromium release assay is performedas described in Example 13E 1.a, where the targets are transduced andnon-transduced EL4 A2/K^(b) (WO 93/15207) and Jurkat A2/K^(b) cells.Non-transduced cell lines are utilized as negative controls. The targetsmay also be peptide coated EL4 A2/K^(b) cells.

(c) Transduction of Human Cells With Vector Construct

Lymphoblastoid cell lines (LCL) are established for each patient byinfecting (transforming) their B-cells with fresh Epstein-Barr virus(EBV) taken from the supernatant of a 3-week-old culture of B95-8, EBVtransformed marmoset leukocytes (ATCC CRL 1612). Three weeks afterEBV-transformation, the LCL are infected with Sindbis vector expressingHBV core or e antigen and G418 resistance. Vector infection of LCL isaccomplished by infecting LCL cells with packaged alphavirus vectorparticles produced from the appropriaste cell line The culture mediumconsists of RPMI 1640, 20% heat inactivated fetal bovine serum (Hyclone,Logan, Utah), 5.0 mM sodium pyruvate and 5.0 mM non-essential aminoacids. Infected LCL cells are selected by adding 800 μg/ml G418. TheJurkat A2/K^(b) cells (L. Sherman, Scripps Institute, San Diego, Calif.)are infected essentially as described for the infection of LCL cells.

(d) Human CTL assays

Human PBMC are separated by Ficoll (Sigma, St. Louis, Mo.) gradientcentrifugation. Specifically, cells are centrifuged at 3,000 rpm at roomtemperature for 5 minutes. The PBMCs are restimulated in vitro withtheir autologous retroviral vector transduced (WO 93/15207) LCL orHLA-matched cells at an effector:target ratio of 10:1 for 10 days.Culture medium consists of RPMI 1640 with prescreened lots of 5%heat-inactivated fetal bovine serum, 1 mM sodium pyruvate and 50 μg/mlgentamycin. The resulting stimulated CTL effectors are tested for CTLactivity using Sindbis vector infected autologous LCL or HLA-matchedcells as targets in the standard chromium release assay, Example 13 1.a.Since most patients have immunity to EBV, the non-transducedEBV-transformed B-cells (LCL) used as negative controls, will also berecognized as targets by EBV-specific CTL along with the transduced LCL.In order to reduce the high background due to killing of labeled targetcells by EBV-specific CTL, it is necessary to add unlabelednon-transduced LCL to labeled target cells at a ratio of 50:1.

2. DETECTION OF HUMORAL IMMUNE RESPONSE

Humoral immune responses in mice specific for HBV core and e antigensare detected by ELISA. The ELISA protocol utilizes 100 μg/well ofrecombinant HBV core and recombinant HBV e antigen (Biogen, Geneva,Switzerland) to coat 96-well plates. Sera from mice immunized withvector expressing HBV core or HBV e antigen are then serially diluted inthe antigen-coated wells and incubated for 1 to 2 hours at roomtemperature. After incubation, a mixture of rabbit anti-mouse IgG1,IgG2a, IgG2b, and IgG3 with equivalent titers is added to the wells.Horseradish peroxidase (“HRP”)-conjugated goat anti-rabbit anti-serum isadded to each well and the samples are incubated for 1 to 2 hours atroom temperature. After incubation, reactivity is visualized by addingthe appropriate substrate. Color will develop in wells that contain IgGantibodies specific for HBV core or HBV e antigen.

3. T CELL PROLIFERATION

Antigen induced T-helper activity resulting from two or three injectionsof Sindbis vector expressing HBV core or e antigen, is measured invitro. Specifically, splenocytes from immunized mice are restimulated invitro at a predetermined ratio with cells expressing HBV core or eantigen or with cells not expressing HBV core or e antigen as a negativecontrol. After five days at 37° C. and 5% CO₂ in RPMI 1640 culturemedium containing 5% FBS, 1.0 mM sodium pyruvate and 10⁻⁵2-mercaptoethanol, the supernatant is tested for IL-2 activity. IL-2 issecreted specifically by T-helper cells stimulated by HBV core or eantigen, and its activity is measured using the CTL clone, CTLL-2 (ATCCTIB 214). Briefly, the CTLL-2 clone is dependent on IL-2 for growth andwill not proliferate in the absence of IL-2. CTLL-2 cells are added toserial dilutions of supernatant test samples in a 96-well plate andincubated at 37° C. and 5%, CO₂ for 3 days. Subsequently, 0.5μCi³H-thymidine is added to the CTLL-2 cells. 0.5Ci ³H-thymidine isincorporated only if the CTTL-2 cells proliferate. After an overnightincubation, cells are harvested using a PHD cell harvester (CambridgeTechnology Inc., Watertown, Mass.) and counted in a Beckman betacounter. The amount of IL-2 in a sample is determined from a standardcurve generated from a recombinant IL-2 standard obtained fromBoehringer Mannheim (Indianaopolis, Ind.).

F. ADMINISTRATION PROTOCOLS

1. MICE

(a) Direct Vector Administration

The mouse system may also be used to evaluate the induction of humoraland cell-mediated immune responses with direct administration of Sindbisvector encoding HBV core or e antigen. Briefly, six- to eight-week-oldfemale C3H/He mice are injected intramuscularly (i.m.) with 0.1 ml ofreconstituted (with sterile deionized, distilled water) orintraperitoneally (ip) with 1.0 ml of lyophilized HBV core or HBV eexpressing Sindbis vector. Two injections are given one week apart.Seven days after the second injection, the animals are sacrificed.Chromium release CTL assays are then performed essentially as describedin Example 13E 1.a.

2. CHIMPANZEE ADMINISTRATION PROTOCOL

The data generated in the mouse system described above is used todetermine the protocol of administration of vector in chimpanzeeschronically infected with hepatitis B virus. Based on the induction ofHBV-specific CTLs in mice, the subjects in chimpanzee trials willreceive four doses of vector encoding core or e antigen at 7 dayintervals given in two successively escalating dosage groups. Controlsubjects will receive a placebo comprised of formulation media. Thedosage will be either 10⁷ or 10⁸ pfu given in four 1.0 ml injectionsi.m. on each injection day. Blood samples will be drawn on days 0, 14,28, 42, 56, 70, and 84 in order to measure serum alanineaminotransferase (ALT) levels, the presence of hepatitis B e antigen,the presence of antibodies directed against the hepatitis B e antigen,serum HBV DNA levels and to assess safety and tolerability of thetreatment. The hepatitis B e antigen and antibodies to HB e antigen isdetected by Abbott HB e rDNA EIA kit (Abbott Laboratories DiagnosticDivision, Chicago, Ill.) and the serum HBV DNA levels is determined bythe Chiron bDNA assay. Efficacy of the induction of CTLs againsthepatitis B core or e antigen can be determined as in Example 13E 1.c.

Based on the safety and efficacy results from the chimpanzee studies,the dosage and inoculation schedule is determined for administration ofthe vector to subjects in human trials. These subjects are monitored forserum ALT levels, presence of HBV e antigen, the presence of antibodiesdirected against the HBV e antigen and serum HBV DNA levels essentiallyas described above. Induction of human CTLs against hepatitis B core ore antigen is determined as in Example 13E 1.c.

G. GENERATION OF ELVIS VECTOR CONSTRUCTS WHICH EXPRESS HBV ANTIGENS FORTHE INDUCTION OF AN IMMUNE RESPONSE

1. CONSTRUCTION OF ELVIS VECTORS EXPRESSING HBVE-C, HBV CORE AND HBV X

Construction of an ELVIS vector expressing the HBV e antigen isaccomplished by digesting the SK⁺HB e-c plasmid with Xho I and Not I torelease the cDNA fragment encoding HBVe-c sequences. The fragment isthen isolated by agarose gel electrophoresis, purified using GENECLEAN™,and inserted into pVGELVIS-SINBV-linker vector , previously prepared bydigestion with Xho I and Not I. This construct is designatedpVGELVIS-HBe.

The HBcore PCR product described previously is digested with Xho I andCla I, isolated by agarose gel electrophoresis, purified usingGENECLEAN™, and ligated into SK+II (Bluescript, Stratagene, Calif.)digested with Xho I and Cla I. This construct is designated SK+HBcore.Construction of the ELVIS vector expressing the HBV core sequence isaccomplished by digesting the SK⁺HBcore plasmid with Xho I and Not I torelease the cDNA fragment encoding HBVcore sequences. The fragment isthen isolated by agarose gel electrophoresis, purified using GENECLEAN™,and inserted into pVGELVIS-SINBV-linker vector, prepared by digestionwith Xho I and Not I. This construct is designated pVGELVIS-HBcore.

Construction of the ELVIS vector expressing the HBV-X antigen sequenceis accomplished by digesting the plasmid SK-X Ag with Xho I and Not I torelease the cDNA fragment encoding HBV-X sequences. The fragment is thenisolated by agarose gel electrophoresis, purified using GENECLEAN, andinserted into the pVGELVIS-SINBV-linker vector, prepared by digestionwith Xho I and Not I. This construct is designated pVGELVIS-HBX.

Any of the above three constructs can be used for selecting vectorinfected cells for the generation of HBV specific CTL targets in thefollowing sections.

2. EXPRESSION OF TRANSFECTED CELLS WITH ELVIS VECTORS

The pVGELVIS-HBe plasmid DNA is isolated and purified, and 2 ug ofpVGELVIS-HBe DNA is complexed with 10 ul of LIPOFECTAMINE™ andtransfected into 2×10⁵ BHK cells contained in 35 mM petri plates. Twodays post-transfection, supernatants and whole cell lysates werecollected and an ELISA assay (see below) was used to determine theamount of expressed HBV-e antigen.

Cell lysates from cells infected by any of the sibling pVGELVIS-HBevectors transfected, are made by washing 1.0×10⁶ cultured cells withPBS, resuspending the cells to a total volume of 600 ul in PBS, andsonicating for two 5-second periods at a setting of 30 in a Bransonsonicator, Model 350 (Fisher, Pittsburgh, Pa.) or by freeze thawingthree times. Lysates are clarified by centrifugation at 10,000 rpm for 5minutes.

Core antigen and precore antigen in cell lysates and secreted e antigenin culture supernatant are assayed using the Abbott HBe, rDNA EIA kit(Abbott Laboratories Diagnostic Division, Chicago, Ill.). Anothersensitive EIA assay for precore antigen in cell lysates and secreted eantigen in culture supernatant is performed using the Incstar ETI-EB kit(Incstar Corporation, Stillwater, Minn.). A standard curve is generatedfrom dilutions of recombinant hepatitis B core and e antigen obtainedfrom Biogen (Geneva, Switzerland).

As shown in FIG. 19, using these procedures, approximately 2 ng/ml HBV eantigen is expressed in the cell lysates and also secreted from BHKcells transfected with different clones of the pVGELVISHBe plasmid.

Characterization of the precore/core and e antigens expressed by vectortransfected cells is performed by immunoprecipitation followed byWestern blot analysis. Specifically, 0.5-1.0 ml of cell lysate in PBS orculture supernatant is mixed with polyclonal rabbit anti-hepatitis Bcore antigen (DAKO Corporation, Carpinteria, Calif.) bound to proteinG-Sepharose (Pharmacia LKB, Uppsala, Sweden) and incubated overnight at4° C. Samples are washed twice in 20 mM Tris-HCl, pH 8.0, 100 mM NaCl,10 mM EDTA and boiled in sample loading buffer with 0.5%2-mercaptoethanol. Proteins are first resolved by SDS polyacrylamide gelelectrophoresis, and then transferred to Immobilon (Millipore Corp.,Bedford, Me.) and probed with the DAKO polyclonal rabbit anti-hepatitiscore B antigen, followed by ¹²⁵I-protein A.

3. TESTING IMMUNE RESPONSE

(a) Administration Protocols

The mouse model system is also used to evaluate the induction of humoraland cell-mediated immune responses following direct administration ofELVIS vector expressing HBV core or e antigen. Briefly, six- toeight-week-old female Balb/c, C57B1/6, C3H/He mice (Charles River,Mass.) and HLA A2.1 transgenic mice (V. Engelhard, Charlottesville, Va.)are injected intramuscularly (i.m.) with, for example, 50 ug or greater,pVGELVIS-HBcore, pVGELVIS-HBVe or pVGELVIS-HBX vector DNA. Twoinjections are given one week apart. Seven or fourteen days after thesecond injection, the animals are sacrificed. Chromium release CTLassays are then performed essentially as described in Example 13E 1.a.Detection of humoral immune responses in mice is performed essentiallyas described in Example 13E 2 and detection of T cell proliferation inmice is performed essentially as described in Example 13E 3.

Example 14 Sindbis Vectors Expressing Viral Proteins for Induction ofthe Immune Response or for Blocking Virus Host Cell Interactions

The following example describes procedures for constructing Sindbisvectors capable of generating an immune response by expressing an HIVviral antigen. Methods are also given to test expression and inductionof an immune response.

Sindbis Vectors Used to Elicit an Immune Response

A. HIV IIIB ENV EXPRESSION VECTOR

A 2.7 Kb Kpn I-Xho I DNA fragment was isolated from the HIV proviralclone BH10-R3 (for sequence, see Ratner et al., Nature 313:277, 1985)and a ˜400 bp Sal I-Kpn I DNA fragment from IIIexE7deltaenv (a Bal31deletion to nt 5496) was ligated into the Sal I site in the plasmid SK⁺.From this clone, a 3.1 kb env DNA fragment (Xho I-Not I) was purifiedand ligated into the previously described Sindbis vectors predigestedwith Xho I and NotI.

B. CREATION OF A PRODUCER CELL LINE WHICH EXPRESSES HIV SPECIFICANTIGENS

To construct a vector producing cell line that expresses the HIV IIIBenv derived from the vector described above, in vitro transcribed RNAtranscripts are transfected in a Sindbis packaging cell line (Example7). Specifically, the Sindbis RNA vector molecules are initiallyproduced by using a SP6 in vitro transcribed RNA polymerase system usedto transcribe from a cDNA Sindbis vector clone encoding the HIV specificsequences. The generated in vitro RNA vector products, are thentransfected into a Sindbis packaging or hopping cell line which leads tothe transient production of infectious vector particles within 24 hours.These vector particles are then collected from the supernatants of thecell line cultures and then filtered through a 0.45 micron filter toavoid cellular contamination. The filtered supernatants are then used toinfect a fresh monolayer of Sindbis packaging cells. Within 24 hours ofinfection, Sindbis vector particles are produced containing positivestranded Sindbis recombinant RNA encoding Sindbis non-structuralproteins and HIV specific sequences.

An alternative configuration of a Sindbis HIV IIIB env vector is apromoter driven cDNA Sindbis construct containing a selectable marker.In this configuration the above-described Xho I to NotI fragmentcontaining the specific HIV IIIB env sequence is placed in a similarcDNA Sindbis vector driven by a constitutive promoter in place of abacteriophage polymerase recognition sequence. Using this configuration,the expression vector plasmids are transfected into the packaging cellline and selected for the drug resistance gene 24 to 48 hourpost-transfection. Resistant colonies are then pooled 14 days later(dependent on the selection marker used) and dilutioned cloned. Severaldilution clones are then propagated, and assayed for highest vectortiter. The highest titer clones are then expanded and stored frozen. Thestored clones are tested for HIV specific protein production and immuneresponse induction.

C. TESTING FOR HIV SPECIFIC PROTEIN PRODUCTION AND AN IMMUNE RESPONSE

Cell lysates from the Sindbis HIV producer cell line are tested for HIVspecific protein production by Western blot analysis. To test theability of the vector to transfer expression in vitro, BHK-21 cells areinfected with filtered supernatant containing viral vector and assayedby Western blot analysis 24 hours post infection. Once proteinexpression has been verified in vivo mouse and primate studies can beperformed to demonstrate the ability of syngeneic cells expressing aforeign antigen after vector treatment to: (a) elicit a CTL response inmice by injecting either infected syngeneic cells or preparations ofinfectious vector; (b) elicit CTL responses in a human in vitro culturesystem; (c) to infect human, chimpanzee and macaque cells, includingprimary cells, so that these can be used to elicit CTL responses and canserve as targets in CTL assays; (d) map immune response epitopes; and(e) elicit and measure CTL responses to other non-HIV antigens such asmouse CMV(MCMV).

1. IMMUNE RESPONSE TO SINDBIS VIRAL VECTOR-ENCODED ANTIGENS

To test the immune response elicited from a cell line transduced with aSindbis HIV IIIB env vector, a murine tumor cell line (B/C10ME)(H-2^(d)) (Patek et al., Cell. Immunol. 72:113, 1982) is infected with arecombinant Sindbis virus carrying the HIV IIIB vector. The HIV envexpressing cell line (B/C10ME-IIIB) was then utilized to stimulate HIVenv-specific CTL in syngeneic (i.e., MHC identical) Balb/c (H-2^(d))mice. Mice are immunized by intraperitoneal injection with B/C10ME-IIIBcells (1×10⁷ cells) and boosted on day 7-14. (Boosting may not berequired.) Responder spleen cell suspensions are prepared from theseimmunized mice and the cells cultured in vitro for 4 days in thepresence of either B/C10ME-IIIB (BCenv) or B/C10ME (BC)mitomycin-C-treated cells at a stimulator:responder cell ratio of 1:50.The effector cells are harvested from these cultures, counted, and mixedwith radiolabled (⁵¹Cr) target cells (i.e., B/C10MEenv-29 or B/C10ME) atvarious effector:target (E:T) cell ratios in a standard 4-5 hour⁵¹Cr-release assay. Following incubation, the microtitre plates arecentrifuged, 100 μl culture supernate is removed, and the amount ofradiolabel released from lysed cells quantitated in a Beckman gammaspectrometer. Target cell lysis was calculated as: % Target Lysis=ExpCPM−SR CPM/MR CPM−SR CPM×100, where experimental counts per minute (ExpCPM) represents effectors plus targets; spontaneous release (SR) CPMrepresents targets alone; and maximum release (MR) CPM representstargets in the presence of 1M HCl.

2. STIMULATION OF AN IMMUNE RESPONSE IN MICE BY DIRECT INJECTION OFRECOMBINANT SINDBIS VECTOR

Experiments are performed to evaluate the ability of recombinant Sindbisviral vectors to induce expression of HIV envelope proteins followingdirect injection in mice. Approximately 10⁴ to 10⁵ (pfu) of recombinantSindbis virus carrying the HIV IIIB env vector construct are injectedtwice (2×) at 3-week intervals either by the intraperitoneal (i.p.) orintramuscular (i.m.) route. This amount of Sindbis virus is determinedto be less than the amount considered to stimulate an immune response.Spleen cells are prepared for CTL approximately 7 to 14 days after thesecond injection of vector.

D. BLOCKING AGENTS DERIVED FROM VIRAL PROTEIN ANALOGUES EXPRESSED FROMRECOMBINANT SINDBIS VECTORS

Many infectious diseases, cancers, autoimmune diseases, and otherdiseases involve the interaction of viral particles with cells, cellswith cells, or cells with factors. In viral infections, viruses commonlyenter cells via receptors on the surface of susceptible cells. Incancers, cells may respond inappropriately or not at all to signals fromother cells or factors. In autoimmune disease, there is inappropriaterecognition of “self” markers. These interactions may be blocked byproducing an analogue to either of the partners in an interaction, invivo.

This blocking action may occur intracellularly, on the cell membrane, orextracellularly. The blocking action of a viral or, in particular, aSindbis vector carrying a gene for a blocking agent, can be mediatedeither from the inside a susceptible cell or by secreting a version ofthe blocking protein to locally block the pathogenic interaction.

In the case of HIV, the two agents of interaction are the gp 120/gp 41envelope protein and the CD4 receptor molecule. Thus, an appropriateblocker would be a vector construct expressing either an HIV envanalogue that blocks HIV entry without causing pathogenic effects, or aCD4 receptor analogue. The CD4 analogue would be secreted and wouldfunction to protect neighboring cells, while the gp 120/gp 41 issecreted or produced only intracellularly so as to protect only thevector-containing cell. It may be advantageous to add humanimmunoglobulin heavy chains or other components to CD4in order toenhance stability or complement lysis. Delivery of a Sindbis vectorencoding such a hybrid-soluble CD4 to a host results in a continuoussupply of a stable hybrid molecule.

Vector particles leading to expression of HIV env analogues may also beconstructed as described above. It will be evident to one skilled in theart which portions are capable of blocking virus adsorption withoutovert pathogenic side effects (Willey et al., J. Virol. 62:139, 1988;Fisher et al., Science 233:655, 1986).

Example 15

A. CONSTRUCTION OF FIV ENV/REV/RRE SINDBIS VECTOR FOR THE INDUCTION OFAN IMMUNE RESPONSE

Sequences encoding the FIV env/rev/RRE gene are amplified and isolatedform plasmid pFIV-14-Petaluma (NIH Research and Reference ReagentProgram, Maryland) using the following primers:

The sense primer sequence has two consecutive Xho I restriction sitesthat are placed at the 5′ end at position 6020 of clone 34F10 (Talbottet al., PNAS 86:5743-5747, 1989): (SEQ. ID NO. 105)

5′-3′: CC CTC GAG CTC GAG GGG TCA CTG AGA AAC TAG AAA AAG AAT TAG

The antisense primer sequence is complementary to a sequence at position9387 of clone 34F10. The 5′ end of the primer has a Not I site (SEQ. IDNO. 106)

5′-3′: CC GCG GCC GC GTA TCT GTG GGA GCC TCA AGG GAG AAC

The PCR product is then placed in the pBluescript KSII+ plasmid(Stratagene, Calif.) and verified by DNA sequencing. This construct isdesignated pBluescript KSII+ FIV env/rev/RRE. The Xho I-Not I fragmentis then excised and inserted into the Sindbis backbone.

Construction of a Sindbis vector expressing the FIV env/rev/RRE sequenceis accomplished by digesting the SK⁺FIV env/rev/RRE plasmid with Xho Iand Not I restriction enzyme sites to release the cDNA fragment encodingFIV env/rev/RRE sequences. The fragment is then isolated by agarose gelelectrophoresis, purified by GENECLEAN™ and inserted into the desiredSindbis vector backbone, prepared by digestion with Xho I and Not I. TheSindbis vectors described in Example 3, are suitable for the insertionof the FIV env/rev/RRE sequences. Such Sindbis vectors include pKSSINBV,pKSSINd1JRsjrc, pKSSINd1JRsjrPC, pKSSINd1JRsjrNP(7582-7601) andpKSSINd1JRsexjr.

The above Sindbis FIV env/rev/RRE expressing vectors may also bemodified to coexpress a selectable drug resistance marker dependent onthe requirements of the experiment or treatment of the vector infectedcells. Any of the above Sindbis FIV env/rev/RRE expression vectorsdescribed may also be designed to coexpress for G418 resistance. This isaccomplished by incorporating an internal ribosomal entry site (Example5) followed by the bacterial neomycin phosphotransferase gene placed 3′of the FIV env/rev/RRE coding sequences and 5′ of the terminal 3′ end ofthe vector using the multiple cloning site of the vector. These G418resistant vector constructs can be used for selecting vector infectedcells for the generation of FIV env/rev/RRE specific CTL targets in thefollowing sections.

B. INFECTION OF FELINE CELLS WITH SINDBIS VECTOR EXPRESSING FIVENV/REV/RRE

The feline kidney cell line (CRFK) is grown in DMEM containing 10% FBS.CRFK cells are infected with the Sindbis vector as described in Examples3 and 7, and used to show vector expression in feline cells usingWestern blot analysis.

C. EXPRESSION OF INFECTED CELLS

Cell lysates from cells infected by any of the FIV env/rev/RREexpressing vectors are made by washing 1.0×10⁷ cultured cells with PBS,resuspending the cells to a total volume of 600 ul in PBS, andsonicating for two 5-second periods at a setting of 30 in a Bransonsonicator, Model 350 (Fisher, Pittsburgh, Pa.) or by freeze thawingthree times. Lysates are clarified by centrifugation at 10,000 rpm for 5minutes.

Proteins are separated according to their molecular weight (MW) by meansof SDS polyacrylamide gel electrophoresis. Proteins are then transferredfrom the gel to a IPVH Immobilon-P membrane (Millipore Corp., Bedford,Mass.). The Hoefer HSI TTE transfer apparatus (Hoefer ScientificInstruments, CA) is used to transfer proteins from the gel to themembrane. The membrane is then probed with either CE4-13B1 or CE3-8,monoclonal antibodies directed against FIV env gp100. The bound antibodyis detected using ¹²⁵I-labeled protein A, which allows visualization ofthe transduced protein by autoradiography.

D. TESTING CELLULAR IMMUNE RESPONSE

1. INBRED MICE

Six- to eight-week-old female Balb/c (H-2d), C57B1/6 (H-2b) and C3H/He(H-2k) mice (Charles River, Mass.) are injected twice intraperitoneally(i.p.) at 1 week intervals with 1×10⁶ pfu of Sindbis FIV env/rev/RREvector. Animals are sacrificed 7 days later and the splenocytes(3×10⁶/ml) cultured in vitro with their respective irradiated (10,000rads) retroviral vector transduced syngeneic cells (WO 94/06921)(6×10⁴/ml) in T-25 flasks (Corning, Corning, N.Y.). These transducedcells include the murine fibroblast cell lines BC10ME (H-2d) (ATCC No.TIB85), B16 (H-2b) and L-M(TK−) (H-2k) (ATCC No. CCL 1.3). These celllines are grown in DMEM containing 4500 mg/L glucose, 584 mg/LL-glutamine (Irvine Scientific, Santa Ana, Calif.) and 10% FBS (Gemini,Calabasas, Calif.). Culture medium consists of RPMI 1640, 5%heat-inactivated fetal bovine serum, 1 mM sodium pyruvate, 50 g/mlgentamycin and 10⁻⁵M 2-mercaptoethanol (Sigma, St. Louis, Mo.). Effectorcells are harvested 4-7 days later and tested using variouseffector:target cell ratios in 96 well microtiter plates (Corning,Corning, N.Y.) in a standard chromium release assay. Targets are theretroviral vector transduced syngeneic cells (WO 94/06921) whereas thenon-transduced syngeneic cell lines are used as negative controls. CTLtargets may also be generated from infecting syngeneic cells with theSindbis FIV env/rev/RRE vector coexpressing the G418 resistance marker.Infected cells are then selected using 800 ug/ml G418 for two weeks.Specifically, Na₂ ⁵¹CrO₄-labeled (Amersham, Arlington Heights, Ill.)(100uCi, 1 hour at 37° C.) target cells (1×10⁴ cells/well) are mixed witheffector cells at various effector to target cell ratios in a finalvolume of 200 μl. Following incubation, 100 ml of culture medium isremoved and analyzed in a Beckman gamma spectrometer (Beckman, Dallas,Tex.). Spontaneous release (SR) is determined as CPM from targets plusmedium and maximum release (MR) is determined as CPM from targets plus1M HCl. Percent target cell lysis is calculated is: [(Effectorcell+target CPM)−(SR)/(MR)−(SR)]×100. Spontaneous release values oftargets are typically 10%-20% of the MR.

For certain CTL assays, the effectors may be in vitro stimulatedmultiple times, for example, on day 8-12 after the primary in vitrostimulation. More specifically, 10⁷ effector cells are mixed with 6×10⁵irradiated (10,000 rads) stimulator cells, and 2×10⁷ irradiated (3,000rads) “filler” cells (prepared as described below) in 10 ml of“complete” RPMI medium. (RPMI containing: 5% heat inactivated FetalBovine Serum. 2 mM L-glutamine, 1 mM sodium pyruvate, 1X non essentialamino acids, and 5×10⁵ M 2-mercaptoethanol). Stimulator cells for invitro stimulation of effector cells are generated from irradiateretroviral vector transduced syngeneic cells. “Filler” cells areprepared from naive syngeneic mouse spleen cells resuspended in RPMI,irradiated with 3,000 rads at room temperature. Splenocytes are washedwith RPMI, centrifuged at 3,000 rpm for 5 minutes at room temperature,and the pellet is resuspended in RPMI. The resuspended cells are treatedwith 1.0 ml tris-ammonium chloride (100 ml of 0.17 M tris base, pH 7.65,plus 900 ml of 0.155 M NH₄Cl; final solution is adjusted to a pH of 7.2)at 37° C. for 3-5 minutes. The secondary in vitro restimulation is thencultured for 5-7 days before testing in a CTL assay. Any subsequentrestimulations are cultured as described above with the addition of 2-10U of recombinant human IL-2 (200 U/ml, catalog #799068, BoehringerMannheim, W. Germany).

2. Felines

Since the vectors are to be utilized for treating felines, an assaydemonstrating immunological efficacy in felines is needed. The followingis a description of the generation of the autologous T-cell lines neededfor restimulator and target cells for the standard ⁵¹Cr release assay(Brown et al., J. Vir. 65:3359-3364, 1991). Briefly, peripheral bloodmononuclear cells (PBMC) are obtained following venipuncture andFicoll-sodium diatrizoate (Histopaque-1077; Sigma, St. Louis, Mo.)density gradient centrifugation. These PBMCs are stimulated by 5 ugm/mlconcanavalin A (Con A, Sigma) for three days, and maintenance in mediumcontaining 25 U/ml human recombinant interleukin-2 (IL-2) (BoehringerMannheim Biochemicals, Indianapolis, Ind.) and 10% bovine T-cell growthfactor (TCGF). Cells are seeded into round bottom 96-well microtiterplates at an average of 1 or 0.3 cells per well with 5×10⁴ irradiated(3,000 rads) autologous PBMC, 10% bovine TCGF, and 25 U/ml of IL-2 in afinal volume of 200 ul of complete RPMI. Complete RPMI consist of RPMI1640 medium containing 10% FBS, 2 mM L-glutamine, 5×10⁵ M2-mercaptoethanol, and 50 ug of gentamycin per ml. Clones are expandedsequentially to 48-well and 24-well plates. After several weeks, cellsare transduced with retroviral vectors expressing FIV env/rev genes (WO94/06921), and selected with G418. Expression of these cell lines aremonitored by Western blot analysis as in Example 15C. Cell linesexpressing high levels of the desired protein function as stimulatorsand targets in a standard ⁵¹Cr release array as in Example 15 D 1.Effector cells are recovered for the CTL assay from the peripheral bloodmononuclear cells (PBMC) obtained following venipuncture andFicoll-sodium diatrizoate density gradient centrifugation.

E. Administration Protocols

Six- to eight-week-old female Balb/C, C57B16 or C3H/He mice are injectedintramuscularly (i.m.) with 0.1 ml of reconstituted (with steriledeionized, distilled water) or intraperitoneally (i.p.) with 1.0 ml oflyophilized FIV env/rev/RRE expressing Sindbis vector. Two injectionsare given one week apart. Seven days after the second injection, theanimals are sacrificed. Chromium release CTL assays are then performedessentially as described in Example 13 D 1.

Felines are also injected intramuscularly (i.m.) with 0.5 ml ofreconstituted (with sterile deionized, distilled water) orintraperitoneally (i.p.) with 2.0 ml of lyophilized FIV env/rev/RREexpressing Sindbis vector. Two injections are given one week apart.Seven days after the second injection, PBMCs are withdrawn for the CTLassay. Chromium release CTL assays are then performed essentially asdescribed in Example 13 D 2.

Example 16 Tissue Specific Expression by Activation of DisabledAlphavirus Vectors Using Tissue Specific Cellular RNA: Construction ofAlphavirus Tumor Specific Expression Vectors for the Treatment ofColorectal Cancer

A. Construction of a Recombinant Sindbis Vector (SIN-CEA) Dependent onthe Expression of the CEA Tumor Marker

As described previously and shown diagrammatically in FIG. 20, thedisabled junction loop out model is constructed with the junction regionof the vector flanked by inverted repeat sequences which are homologousto the RNA of choice. In this example, sequences from the CEA tumorantigen cDNA (Beauchemin et al., Molec. and Cell. Biol. 7:3221, 1987)are used in the inverted repeats. To construct a CEA RNA responsiveSindbis vector, the junction region is preceded by two CEA anti-sensesequence domains (A¹ and B¹) separated by a six base pair hinge domain.A single twenty base pair CEA sense sequence (A2), which iscomplementary to A1, is placed at the 3′ end of the junction region. Inchoosing the correct A1 and B1 antisense sequences, the only tworequirements are that they be specific for the target RNA sequence andthat the anti-sense sequences hybridize to two RNA sequence domainsseparated by three nucleotides. This three nucleotide gap will serve asa hinge domain for the polymerase to hop and switch reading strandsbridging the non-structural protein domain of the vector to the junctionregion of the vector (FIG. 5). To construct such a configuration, twoologonucleotides are synthesized complementing each other to create afragment insert containing convenient restriction enzyme sites at theenzyme 5′ and 3′ ends. The oligonucleotide fragment insert is thenligated into the Sindbis vector between the disabled junction region andthe multiple cloning sites of the Sindbis vector. The senseoligonucleotide strand, from 5′ to 3′, should contain an Apa Irestriction site, followed by the A1 anti-sense domain, a six bp hingedomain, a B1 anti-sense domain, a synthetic junction region domain, andthe A2 sense domain, followed by a Xho I restriction enzyme site. Thefollowing oligonucleotide sequence is used to design a CEA RNAresponsive Sindbis vector. The nucleotide number sequence is obtainedfrom Beauchemin et al., Molec. and Cell Biol. 7:3221, 1987.

5′-3′ CEA sense strand:                CEA618                         CEA 589       ApaI    *------------------------------------* CGC GC  G GGC  CCT GT  G ACAT  TG AAT  AGA GT  G AGG G TC CTG TTG GG (SEQ. ID NO. 107)     CEA651                          CEA 622    *--------------------------------------*    *   Synthetic A AAGG  TT TCA  CAT TT  G TAG C  TT GCT  GTG TC  A TTG C  GA TCT CTA CG (SEQ.ID NO. 108)                      CEA 599           CEA 618  JunctionCore   *   *------------------------*   Xho I G TGG T  CC TAA  ATA GT  TCAC T  CT ATT  CAA TG  T CAC A  CT CGA GCC GG (SEQ. ID NO. 109)

The 5′-3′ CEA anti-sense strand is complementary to the aboveoligonucleotide. After both oligonucleotides are synthesized, theoligonucleotides are mixed together in the presence of 10 mM Mg, heatedto 100° C. for 5 minutes and cooled slowly to room temperature. Theoligonucleotide pair is then digested with the Apa I and Xho Irestriction enzymes, mixed and ligated at a 25:1 molar ratio of insertto plasmid, pCMV-SIN or pMET-SIN predigested with the same enzymes.These constructs are designated pMCV/SIN-CEA and pMET/SIN-CEA,respectively.

Construction of a SIN-CEA Vector and Producer Cell Line Expressing GammaInterferon (SIN-CEA/IFN)

The human gamma interferon gene is subcloned from the retroviral vectorplasmid pHu-IFN-γ (Howard et al., Ann N.Y. Acad. Sci. 716:167-187, 1994)by digesting with Xho I and Cla I. The resulting 500 bp fragmentcontaining the coding sequences of γ-IFN is isolated from a 1% agarosegel.

Alternatively, the human γ-IFN cDNA is derived from RNA isolated fromPHA-stimulated Jurkat T cells by guanidinium thiocyanate extractionfollowed by ultracentrifugation through a CaCl gradient. The RNA (Sigma,St. Louis, Mo.) is then reverse-transcribed in vitro and a gene-specificoligonucleotide pair is used to amplify γ-IFN cDNA by polymerase chainreaction using Taq polymerase. The PCR DNA was repaired with T4 DNApolymerase and Klenow and cloned into the Hinc II site of SK⁺ plasmid(Stratagene, San Diego, Calif.) treated with CIAP. In the senseorientation, the 5′ end of the cDNA is adjacent to the Xho I site of theSK⁺ polylinker and the 3′ end adjacent to the NotI site. The 512 basepair fragment encoding the human γ-FIN molecular is placed into the XhoI/NoyI site of either the pCMV/SIN-CEA or pMET/SIN-CEA vectors. Thesenew plasmids are designated pCMV/SIN-CEA/IFNγ or pMET/SIN-CEA/IFN-γ,respectively.

B. Construction of a SIN-CEA Vector and Producer Cell Line ExpressingThymidine Kinase (SIN-CEA/TK)

A PCR amplified product containing the cDNA clone of the herpes simplexthymidine kinase (“HSVTK”), flanked with 5′ Xho I and 3′ NotIrestriction enzyme sites is obtained using the pHS1TK3KB (Mcknight etal., Nuc. Acids Res. 8:5949, 1980) clone as target DNA. The sequencesfor the primers used for the PCR amplification are obtained frompublished sequences (Wagner et al., PNAS 78:1442, 1981). The 1,260 basepair amplified product is then digested with Xho I and NotI digestedinto the Xho I/NotI site of either the pCMV/SIN-CEA or pMET/SIN-CEAvectors. These new plasmids are designated pCMV/SIN-CEA/HSVTK orpMET/SIN-CEA/HSVTK, respectively.

C. Creation of CEA RNA Dependent Sindbis Vector Producer Cell Lines

Unlike the previous examples of creating producer cell lines (Example7), it may be that only a single round of gene transfer into thepackaging cell line is possible by vector transfection. Since thesevectors will be disabled and prevented in the synthesis of full genomicvectors, re-infection of a fresh layer of Sindbis packaging cell lineswill end in an aborted infection since these vectors are now dependenton the presence of the CEA RNA to become active. Higher titers may beachieved by dilution cloning transfected producer cell lines using theRT-PCR technique.

Example 17 Replacement Gene Therapy Using Recombinant Alphavirus Vectors

The following example describes the construction of alphavirus vectorscapable of generating a therapeutic protein.

A. Construction of a Sindbis Factor VIII Vector

Hemophilia A disease is characterized by the absence of Factor VIII, ablood plasma coagulating factor. Approximately 1 in 20,000 males havehemophilia A in which the disease state is presented as a bleedingdisorder, due to the inability of affected individuals to complete theblood clotting cascade.

The treatment of individuals with hemophilia A is replacement with theFactor VIII protein. The only source for human Factor VIII is humanplasma. In order to process human plasma for Factor VIII purification,human donor samples are pooled in lots of over 1000 donors. Due to theinstability of the Factor VIII protein, the resulting pharmaceuticalproducts are highly impure, with an estimated purity by weight ofapproximately 0.04%. In addition, there is a serious threat of suchinfectious diseases as hepatitis B virus and the Human ImmunodeficiencyVirus, among others, which contaminate the blood supply and can thus bepotentially co-purified with the Factor VIII protein.

The Factor VIII cDNA clone is approximately 8,000 bps. Insertion of theFactor VIII cDNA into pKSSINBV yields a vector/heterologous gene genomicsize of approximately 15,830 bps. If the packaging of this large vectorRNA into particles is inefficient, the size of the insert can bedecreased further by eliminating the “B-domain” of the Factor VIIIinsert. It has been that the Factor VIII B-domain region can be removedfrom the cDNA without affecting the functionality of the subsequentlyexpressed protein.

A Sindbis-Factor VIII vector is constructed as follows. Factor VIII cDNAis obtained from clone pSP64-VIII, an ATCC clone under the accessionnumber 39812, containing a cDNA encoding the full-length human protein.pSP64-VIII is digested with Sal I, the termini are blunted with T4 DNApolymerase and 50 uM of each dNTP, and the ca. 7700 bp. fragment iselectrophoresed in a 1% agarose/TBE gel and purified with GENECLEAN™.The Factor VIII cDNA containing blunt ends is then ligated intopKSII3′SIN (Example 3), prepared by digestion with Hinc II, treated withCIAP, and purified from a 1% agarose gel. The plasmid is known aspF83′SIN.

For insertion of Factor VIII into the various Sindbis vectors describedin Example 3, plasmid pF83′SIN is digested with Xho I and a limited SacI digest, and the resulting 7,850 bp fragment is isolated from a 0.7%agarose/TBE gel. This Factor VIII-3′SIN fragment is then inserted intoeach of the vectors listed below. Prior to insertion of this fragmentthe plasmids are prepared by digestion with Xho I and Sac I, treatedwith CIAP, isolated by 1% agarose/TBE gel electrophoresis, and purifiedwith GENECLEAN™:

Vector Functional Junction Region (+/−) pKSSINBV + pKSSINd1JRsjrc +pKSSINd1JRsjrPC + pKSSINd1JRsjrNP(7,582-7,601) + pKSSINd1JRsexjr +

Following insertion of the Factor VIII cDNA, these vectors aredesignated:

pKSSINBVF8

pKSSINd1JRsjrcF8

pKSSINd1JRsjrPCF8

pKSSINd1JRsjrNP(7,582-76,601)F8

pKSSINd1JRsexjrF8

respectively.

Packaging of the Factor VIII cDNA containing vectors is accomplished bythe transfection of packaging cell lines (described in Example 7) within vitro transcribed vector/Factor VIII RNA. The efficiency of packagingis determined by measuring the level of Factor VIII expression in cellsinfected with the packaged vector and compared to similar experimentsperformed with the pKSSIN-luc vector described in Example 3.

B. Construction of a Glucocerebrosidase Sindbis Vector

Gaucher disease is a genetic disorder that is characterized by thedeficiency of the enzyme glucocerebrosidase. This enzyme deficiencyleads to the accumulation of glucocerebroside in the lysosomes of allcells in the body. However, the disease phenotype is manifested only inthe macrophages, except in the very rare neurophatic forms of thedisease. The disease usually leads to enlargement of the liver andspleen and lesions in the bones. (For a review, see Science 256:794,1992, and The Metabolic Basis of Inherited Disease, 6th., Scriver etal., vol. 2, p. 1677.)

A glucocerebrosidase Sindbis vector is constructed as follows. Briefly,a glucocerebrosidase (GC) cDNA clone containing a Xho I restrictionenzyme site 5′ and 3′ of the cDNA coding sequence is first generated.The clone is generated by digesting pMFG-GC (Ohashi et al., PNAS89:11332, 1992) the Nco I, blunt-ending the termini with T4 DNApolymerase and dNTPs, ligating with Xho I linkers, and purifying the GCgene from a 1% agarose gel. The GC fragment is subsequently digestedwith Xho I and ligated with the desired Sindbis vector (for example,pKSSINBV) that has also been digested with Xho I. Packaging of theSindbis-glucocerebrosidase vector is accomplished by introduction ofvector RNA (for example, transfection of in vitro transcribed RNA) intoany of the packaging cell lines described in Example 5.

Both the Sindbis Factor VIII and the Sindbis Glucocerebrosidase vectorsare also readily convertible to plasmid DNA based-vectors which initiatevector replication and heterologous gene expression for use in directdelivery or the establishment of vector producer cell lines (seeExamples 3 and 7).

Example 18 Inhibition of Human Papilloma Virus Pathogenicity bySequence-Specific Antisense or Ribozyme Molecules Expressed from SindbisVirus Vectors

To date, more than sixty types of human papilloma viruses (HPV), whichhave a pronounced tropism for cells of epithelial origin, have beenisolated and characterized. Among the HPV group are a substantial numberof types which infect the human anogenital tract. This group of HPVs canbe further subdivided into types which are associated with benign orwith malignant proliferation of the anogenital tract.

There are between 13,000 and 20,000 cervical cancer deaths per year inthe U.S. In developing countries, cervical cancer is the most frequentmalignancy, and in developed countries cervical cancer ranks behindbreast, lung, uterus, and ovarian cancers. One statistic whichespecially supports the notion that anogenital proliferation is agrowing health problem is that medical consultations for genital wartsincreased from 169,000 in 1966 to greater than 2 million in 1988.

Several lines of evidence exist which link HPV to the pathogenesis ofcervical proliferative disease. A distinct subset of types, so called‘low risk HPVs’, are associated with benign proliferative states of thecervix (e.g., HPV 6, 11, 43, 44), while another subset of types, the‘high risk HPVs’, are associated with lesions which may progress to themalignant state (e.g., HPV 16, 18, 31, 33, 35, etc.). Approximately 95%of cervical tumors contain HPV, with HPV type 16 or 18 DNA being foundin about 70% of them.

The frequency of HPV in the young sexually active female populationappears to be quite high. Indeed, in a recent study of 454 collegewomen, 213, or 46% were HPV positive. Among the HPV positive group, 3%were HPV 6/11 positive, and 14% were HPV 16/18 positive. Of these 454women, 33 (7.3%) had abnormal cervical proliferation, as determined bycytology.

With regard to the design of antisense and ribozyme therapeutic agentstargeted to HPV, there are important parameters to consider relating tothe HPV types to target (i.e., types associated with condylomaacuminatum or types associated with malignant cervical proliferation)and HPV expressed genes to target, including but not limited to, HPVgenes E2, E6, or E7.

In general, the expression of HPV genes is defined temporally in twophases, early (E) genes expressed prior to viral DNA replication, andlate (L) genes expressed after viral DNA replication. There are 7 earlyenzymatic HPV genes, and 2 late structural HPV genes.

Based on the discussion presented above, antisense/ribozyme therapeuticsdirected towards the HPV 6/11 groups may be constructed which target theviral E2 gene. It seems possible that the E2 gene target may beprecarious with regard to the HPV 16/18 group, by a mechanism of drivingintegration of the virus through of E2 protein expression. Thus, itseems that the E6/E7 genes in HPV types 16/18 should be targeteddirectly.

Described below is the construction of antisense and ribozymetherapeutics into Sindbis virus vectors (described in Example 2)specific for HPV type 16 E6 and E7 RNA. Insertion of the HPV antisenseand ribozyme moieties is between the Cla I and Xba I sites of theSindbis vector.

A. Construction of an HPV 16 E6/E7 Antisense Therapeutic

The HPV 16 viral genomic clone, pHPV-16 (ATCC number 45113) is used as atemplate in a PCR reaction for the amplification of specific sequencesfrom the viral E6/E7 genes. The HPV 16 antisense moiety is firstinserted into the plasmid vector pKSII⁺; removal of the antisensetherapeutic from the plasmid vector and insertion into the variousSindbis vector backbones is accomplished via the unique antisense moietyterminal Cla I and Xba I restriction endonuclease sites. Amplificationof a portion of the HPV 16 E6/E7 genes is accomplished with the primerpair shown below:

Forward primer (buffer sequence/Xba I site/HPV 16 nucleotides 201-222):

TATATTCTAGAGCAAGCAACAGTTACTGCGACG (SEQ ID NO. 110)

Reverse primer (buffer sequence/Cla I HPV 16 nucleotides 759-738):

TATATATCGATCCGAAGCGTAGAGTCACACTTG (SEQ ID NO. 111)

In addition to the HPV 16 E6/E7 complementary sequences, both primerscontain a five nucleotide ‘buffer sequences’ at their 5′ ends forefficient enzyme digestion for the PCR amplification products.Generation of the HPV 16 amplicon with the primers shown above isaccomplished with the PCR protocol described in Example 4. It has beenshown previously that the E6/E7 mRNA in infected cervical epithelia ispresent in three forms, unspliced and two spliced alternatives (E6* andE6**), one in which nucleotides 226-525 of E6 are not present in themature message (Smotkin et al., J. Virol 63:1441-1447, 1989). The regionof complementary between the antisense moiety described here and the HPV16 genome is viral nucleotides 201-759. Thus the antisense moiety willbe able to bind to and inhibit the translation of the E6E7 unsplicedmessage and the spliced E6* and E6** spliced messages.

The HPV 16 E6/E7 580 bp amplicon product is first purified withGENECLEAN™, digested with the restriction enzymes Cla I and Xba I, andelectrophoresed on a 1% agarose/TBE gel. The 568 bp band is then excisedfrom the gel, the DNA purified with GENECLEAN™ and ligated into thepKSII⁺ plasmid prepared by digestion with Cla I and Xba I, treatmentwith CIAP, and treatment with GENECLEAN™. This plasmid is known aspKSaHPV16E6/E7.

B. Construction of HPV 16 E6/E7 Hairpin Ribozyme Therapeutics

In order to efficiently inhibit the expression of HPV 16 E6/E7 proteins,a hairpin ribozyme (HRBZ) with target specificities to E6 mRNA isconstructed. The HPV 16 ribozyme moiety is first inserted into theplasmid vector pKSII⁺; removal of the ribozyme therapeutic from theplasmid vector and insertion into the various Sindbis vector backbonesis accomplished via the unique ribozyme moiety terminal Cla I and Xba Irestriction endonuclease sites.

The HRBZ is homologous to the HPV 16 E6 RNA (nts 414-431) shown below:

TTAACTGTCAAAAGCCAC (SEQ ID NO. 112)

The HRBZ is designated to cleave after the T residue in the TCTC hairpinribozyme loop 5 substrate motif, shown underlined above. Followingcleavage, the HRBZ is recycled and able to hybridize to, and cleave,another unspliced E6/E7 mRNA or the E6* spliced mRNA molecule.

Double-stranded HRBZ as defined previously (Hampel et al., Nucleic AcidsResearch 18:299-304, 1990), containing a 4 base ‘tetraloop’ 3 and anextended helix 4, with specificity for the HPV 16 E6 RNA shown above, ischemically synthesized and includes both the 5′ and 3′ ends,respectively, Cla I and Xba I sites. The sequence of the chemicallysynthesized HPV 16 E6 HRBZ strands are shown below:

HPV 16 E6 HRBZ, sense strand (5′→3′):

5′-CGATGTGGCTTTTAGATGTTAAACCAGAGAAACACACGGACTTCGGTCCGTGGTATATTAGCTGGTAT-3′ (SEQ. ID NO. 113)

HP 16 E6 HBRZ, antisense strand (5′→3′):

5′-CTAGATACCAGCTAATATACCACGGACCGAAGTCCGTGTGTTTCTCTGGTTTAACATCTAAAAGCCACAT-3′ (SEQ. ID NO. 114)

In order to form the double-stranded HPV 16 E6 specific HRBZ with Cla Iand Xba I cohesive ends, equal amounts of the oligonucleotides are mixedtogether in 10 mM Mg²⁺, heated at 95° C. for 5 minutes, then cooledslowly to room temperature to allow the strands to anneal.

The double-stranded HPV 16 E6 HRBZ with Cla I and Xba I cohesive ends isfirst ligated into the pKSII⁺ plasmid vector, prepared by digestion withCla I and Xba I, treatment with CIAP, and treatment with GENECLEAN™. Theplasmid is known as pKSHPV16E6HRBZ.

The HPV 16 antisense and hairpin ribozyme moieties are liberated fromtheir plasmid vectors, pKSaHPV16E6/E7 and pKSHPV16E6HRBZ, respectively,by digestion with Cla I and Xba I, purification by agaroseelectrophoresis and GENECLEAN™, and insertion into the desired vectorbackbone, prepared by digestion with Cla I and Xba I, and treatment withCIAP. Several possible Sindbis vectors some of which are shown below,and those detailed construction is described in Example 2, are suitablefor the insertion of the HPV 16 antisense and ribozyme therapeuticmoieties:

Vector Functional Junction Region (+/−) pKSSINBV + pKSSINBVd1JR −pKSSINd1JRsjrc + pKSSINd1JRsjrPC + pKSSINd1JRSjrNP(7582-7601) +pKSSINdlJRsexjr +

Since the antisense and ribozyme therapeutic operate at the level ofRNA, it is not necessary that the vectors containing these moietiescontain a functional junction region. That is, translation of the regioncorresponding to the Sindbis structural proteins occurs only fromsubgenomic RNA. However, because translation of the antisense andhairpin ribozyme therapeutic is not an issue, these moieties will exerttheir affect from the level of positive stranded Sindbis genomic vectorRNA.

On the other hand, it may be desired to administer repeated doses to anindividual; thus the antisense and hairpin palliative would be inserteddownstream of the adenovirus E3 or human cytomegalovirus H301 genes,which down-regulate the expression of MHC class I molecules in infectedcells. Insertion of the antisense and hairpin palliatives isaccomplished in the vectors from Examples 3 and 4 shown below, betweenthe Cla I and Xba I sites:

Vector Functional Junction Region (+/−) pKSSINd1JRsjrcAdE3 +pKSSINd1JRsjrcH301 +

Subgenomic mRNA is synthesized in these vectors, which serves as atranslational template for the Ad E3 and CMV H301 genes. Thus, in theseconstructions, functional HPV 16 antisense and hairpin ribozymepalliatives will be present on the levels of both subgenomic andpositive stranded genomic Sindbis vector RNA.

Further, the HPV 16 antisense and hairpin ribozyme palliatives can beinserted downstream of a heterologous gene inserted into the describedSindbis vectors. For example, one could insert the HPV 16 antisense andhairpin ribozyme palliatives downstream of a heterologous gene codingfor an immunogenic epitope of HPV 16 form, for example, the E6/E7 or L1proteins. In these vectors, it would not be desired to include theimmunoregulatory Ad E3 or CMV H301 genes.

Expression of the E6/E7 genes during infection with both the high- andlow-risk HPV groups is required for proliferation of the cervicalepithelium. The HPV E7 protein from all HPV types tested forms a complexwith the retinoblastoma protein, and the E6 protein from HPV types 16and 18 associates with and degrades the cellular p53 protein. The p53and retinoblastoma cellular gene products are involved in the growthcontrol of the cell, and altering the expressing or function of theseproteins can release the growth control in affected cells. Thus, anantisense or ribozyme therapeutic agent to both HPV groups should eitherdirectly or ultimately diminish the expression of one or both of thesegenes. Expression of the E6/E7 genes is trans-activated by the viral E2protein. However, by utilizing an alternative splicing strategy, the E2protein can also act as a trans-repressor. Integration of the oncogenicHPV types occurs in the viral E2 region and abrogates the expression ofthe E2 protein. Integration by the oncogenic HPV types appears to be apivotal event in the frank induction and/or maintenance of cervicalcarcinoma. This event results in the constitutive expression of theE6/E7 genes. In the integrated state, expression of the E6/E7 genes istrans-activated by factors present in infected keratinocytes. Theinactivation of the viral E2 control mechanism in response to thecellular keratinocyte factor activation of E6/E7 expression might be acritical event in viral integration.

Example 19 Inhibition of Human Interferon a Expression in Infected Cellsby Sequence: Specific Ribozyme Molecules Expressed from Sindbis VirusVectors

Interferons (IFNs) comprise a family of small proteins which effect awide range of biological activities in the mammalian cell, including theexpression of MHC antigens, the expression of several genes whichmodulate cell growth control, and the resistance to viral infections(Pestka et al., Ann. Rev. Biochem. 56:727-777, 1987). Of the threeclasses of IFNs, α, β, and γ-IFN, α-IFN, or leukocyte interferon, has akey role in limiting viral replication in the infected cell.

The antiviral effects of IFN-α are associated with the induction of twocellular enzymes which inhibit the viral lifecycle in the infected cell.One enzyme is a double-stranded RNA dependent 68-kDa protein kinase thatcatalyzes the phosphorylation of the α subunit of the protein synthesisinitiation factor eIF-2. The second enzyme induced by IFN- is2′,5′-oligoadenylate synthetase (2′,5′-OAS), which in the presence ofdouble-stranded RNA activates the latent endonuclease, RNase L, which isresponsible for degradation of viral and cellular RNAs (Johnston andTorrence, Interferons 3:189-298, Friedman (ed.), Elsevier SciencePublishers, B. V., Amsterdam, 1984).

Because their replication strategy includes a double-stranded RNAintermediate, the RNA viruses in particular are strong inducers ofinterferon. With regard to Sindbis virus, double-stranded RNA moleculesare present during the replication of both positive- andnegative-stranded genome length molecules, and during the transcriptionof subgenomic mRNA. It has been demonstrated that infection of cellswith Sindbis virus results in the induction of interferon (Saito, J.Interferon Res. 9:23-24, 1989).

In applications where extended expression of the therapeutic palliativeis desired, expression of IFN in the infected cell is inhibited byinclusion of a hairpin ribozyme with specificity for IFN-α mRNA in theSindbis vector. Inhibition of IFN-expression thus mitigates induction ofthe cascade of cellular proteins, including the eIF-2 protein kinase and2′,5′-OAS, which inhibit the extent to which virus can replicate in theinfected cell. Prolonged expression of the therapeutic palliativewithout induction of an immune response targeted towards the vectorinfected cell is desired in all applications other than antigenpresentation and includes, for example, systemic protein production,antisense and ribozyme, and accessory molecules.

A. Construction of a Hairpin Ribozyme with Targeted Specificity forInterferon A mRNA

In order to efficiently inhibit the expression of interferon α proteinin cells infected with Sindbis vectors, a hairpin ribozyme (HRBZ) withtarget specificity for interferon α mRNA is constructed. The IFN-αribozyme moiety is first inserted into the plasmid vector pKSII⁺(Stratagene, La Jolla, Calif.): removed of the ribozyme therapeutic fromthe plasmid vector and insertion into the various Sindbis vectorbackbones is accomplished via the unique ribozyme moiety terminal Cla Iand Xba I restriction endonuclease sites.

The HRBZ is homologous to nucleotides 1026-1041 of the human interferonalpha gene IFN-alpha 4b shown below, and to all IFN-α genes sequenced,including 5, 6, 7, 8, and 14, but not gene 16 (Henco et al., J. Mol.Biol. 185:227-260, 1985):

5′-TCT CTG TCC TCC ATG A (SEQ. ID NO. 120)

The HRBZ is designed to cleave after the T residue in the TGTC hairpinribozyme loop 5 substrate motif, shown underlined above. Followingcleavage, the HRBZ is recycled and able to hybridize to, and cleave,another IFN-a mRNA molecule.

Double-stranded HRBZ as defined previously (Hampel et al., Nucleic AcidsResearch 18:299-304, 1990), containing a 4 base tetraloop 3 and anextended helix 4, with specificity for the IFN-a mRNA shown above, ischemically synthesized and includes at the 5′ and 3′ ends, respectively.Cla I and Xba I sites. The sequence of the chemically synthesized IFN-aHRBZ strands are shown below:

IFN-α HRBZ, sense strand (5′ to 3′):

TCG AGT CAT GGA GAG AGG AGA ACC AGA GAA ACA CAC GGA CTT CGG TCC GTG GTATAT TAC CTG GAT (SEQ. ID NO. 121)

IFN-α HBRZ, antisense strand (5′ to 3′):

CGA TCC AGG TAA TAT ACC ACG GAC CGA AGT CCG TGT GTT T CTCTG GTT C TC CTCTCT CCA TGA C (SEQ. ID No. 122)

In order to form the double-stranded IFN-α specific HRBZ with Cla I andXba I cohesive ends, equal amounts of the oligonucleotides are mixedtogether in 10 mM Mg²⁺, heated at 95° C. for 5 minutes, then cooledslowly to room temperature to allow the strands to anneal.

The double-stranded IFN-α HRBZ with Cla I and Xba I cohesive ends isfirst ligated into the pKSII⁺ plasmid vector, prepared by digestion withCla I and Xba I, treatment with CIAP, and treatment with GENECLEAN™.This plasmid is known as pKSIFNαHRBZ.

The INF-α hairpin ribozyme moiety is liberated from the pKSIFNaHRBZplasmid by digestion with Cla I and Xba I, purification by 2%Nu-Sieve/1% agarose electrophoresis and GENECLEAN™, and insertion intothe desired vector backbone, prepared by digestion with Cla I and Xba I,and treatment with CIAP. Several possible Sindbis vectors some of whichare shown below, and whose detailed construction is described inExamples 2, 3, and 4 are suitable for the insertion of the IFN-α hairpinribozyme moiety:

Vector Functional Junction Region (+/−) pKSSINBV + pKSSINBVd1JR −pKSSINd1JRsjrc + pKSSINd1JRsjrPC + pKSSINd1JRsjrNP(7582-7601) +pKSSINd1JRsexjr + pKSSINd1JRsjrcAdE3 + pKSSINd1JRsjrcH301 +

Since the ribozyme activity operates at the level of RNA, it is notnecessary that this region is expressed as a portion subgenomic mRNA.However, when placed downstream of a functional junction region, thelevel of ribozyme synthesized is much greater and perhaps more effectivein cleaving the IFN-α RNA target.

Further, in some applications, for example systemic expression ofprotein, multiple dose administration to an individual is required. Inthese applications, prolonged expression of the therapeutic palliativewithout induction of an immune response targeted towards the vectorinfected cell is desired. In this configuration, the IFN-αHRBZ moietycould be inserted upstream of the adenovirus E3 or human cytomegalovirusH301 genes, which down-regulate the expression of MHC class I moleculesin infected cells. Following the gene which modulates MHC class Iexpression is, consecutively, an IRES element selected from among thegroup described in Example 5, and the therapeutic palliative. Orderedinsertion of the hairpin ribozyme, Ad E3 or CMV H301, IRES, andheterologous gene of interest components along the multiple cloningsequence located in the vector between the vector junction region and 3′end is accomplished by modification with the appropriate restrictionenzyme recognition sites of the compound 5′ and 3′ ends. In theseconstructions, functional IFN-a hairpin ribozyme palliatives will bepresent at the level of both subgenomic and positive stranded genomicSindbis vector RNA.

Example 20 Ex vivo and in vivo treatment of Human Cancers byAdministration of Recombinant Alphavirus Vector Particles or AlphavirusPlasmid DNA Vectors which Express Cytokines, Cytokine Receptors, or DrugPotentiators

A. Vector Constructions

1. Gamma Interferon

Murine gamma interferon is subcloned from the retroviral vector plasmidpMu-γIFN (Howard et al., Ann. N.Y. Acad. Sci. 716:167-187, 1994) bydigesting with Cla I and making the termini blunt by Klenow enzyme anddNTPs. After heat inactivation of the Klenow enzyme, the vector isdigested with Xho I. The resulting 800 bp fragment containing the codingsequences of gamma interferon is isolated from a 1% agarose gel.pKSSINBV (Example 3) is digested with Xho I and Stu I, and the vector ispurified by GENECLEAN™ and ligated with the gamma interferon insert. Theresulting vector construction is known as pKSSINγMu. The human gammainterferon gene (Howard et al., supra) is similarly inserted intopKSSINBV using the same strategy. The resulting vector construct isknown as pKKSINγHu. The interferon expressing Sindbis vectors are thenpackaged into vector particles. This is accomplished by introducing RNAfrom these vectors into a packaging cell line as described in Example 7.

The mouse and human interferon genes are also cloned intopVGELVISSINBV-linker (see Example 3). Briefly, pVGELVISSINBV-linker isfirst digested with Asc I and the termini made blunt by the addition ofKlenow enzyme and dNTPs. The Klenow is heat inactivated and the vectoris subsequently digested with Xho I. This vector is purified byGENECLEAN™ and ligated to the gamma interferon inserts prepared asdescribed above. The resulting vectors are described pVGELVIS-γMu andpVGELVIS-γHu, respectively.

2. Interferon-2

The human IL-2 gene is cloned by PCR amplification into the KT-3retroviral backbone (Howard et al., Ann N.Y. Acad. Sci. 716:167-187,1994). The source for the IL-2 gene is a pBR322 based plasmid whichcontains the IL-2 cDNA (ATCC #61391). The cDNA is PCR amplified using astandard three-temperature protocol as described in Example 3. The 5′primer is the sense sequence of the hIL-2 gene complimentary to the 5′coding region beginning at the ATG start codon. Additionally, a Xho Isite is built into the 5′ end of the primer sequence.

5′hIL-2 (SEQ. ID NO. 123)

5′-GCCTCGAGACAATGTACAGGATGCAACTCCTGTCT

The 3′ primer is an antisense sequence of the hIL-2 gene complementaryto the 3′ coding region ending at the TAA stop codon. Additionally, aCla I site is built into the 5′ end of the primer sequence.

3′ hIL-2 (SEQ. ID NO. 124)

5′-GAATCGATTTATCAAGTCAGTGTTGGAGATGATGCT

The PCR amplicon is purified in a 1% agarose gel. To place the IL-2 genein the KT-3 retroviral backbone, pMu-IFN is digested with Xho I and ClaI to remove the interferon gene. After treatment with phosphatase, thevector is purified in a 1% agarose gel. The vector and IL-2 insert areligated and transformed using standard procedures, and recombinantclones are screened by restriction enzyme analysis. The resulting vectoris designated pKThIL-2.

Human IL-2 is subcloned from the retroviral vector pKThIL2, into thepKSSINBV vector, using the same strategy employed for murine gammainterferon. The resulting vector construction is known as pKSSIN-huIL-2.The human IL-2 gene is also cloned into pVGELVISSINBV-linker asdescribed above for the gamma interferon genes. The resulting constructis designated pVGEL VIS-IL-2.

3. HSV-TK

The coding region and transcriptional termination signals of HSV-1thymidine kinase gene (HSV-TK) are isolated as a 1.8 kb Bgl II/Pvu IIfragment from plasmid 322TK (McKnight et al., Nuc. Acids Res. 8:5949,1980) cloned into pBR 322 (ATCC No. 31344). The ends are made blunt bythe addition of Klenow enzyme and dNTPs. The 1.8 kb fragment is isolatedon a 1% agarose gel and ligated to pKS SINBV which had been previouslydigested with Stu I, phosphatased and gel purified. This construct isknown as pKSSINBV-TK. For use is physical gene transfer experiments, theTK gene is similarly cloned into pVGEL VIS-SINBV-linker. The vector isprepared by digestion with Pml I, phosphatase treatment and isolated ona 1% agarose gel. This vector construct is known as pVGEL VISBV-TK.

B. Administration

Any of the above-described vector constructs may be utilized along withpackaging cell lines described in Example 7, in order to producerecombinant alphavirus particles suitable for administration to humansor animals (either directly or indirectly), or for infecting targetcells. Such vector constructs may also introduced directly into targetcells as a “naked” DNA molecule, as a DNA complex with various liposomeformulations, or as a DNA ligand complex including the alphavirus DNAvector molecule (e.g., along with the polycation compound such aspolylysine, a receptor specific ligand, or a psoralen inactivated virussuch as Sendai or Adenovirus).

This aspect of the invention relates to pharmaceutical compositionscomprising alphavirus vector constructs, recombinant alphavirusparticles, or eukaryotic layered vector initiation systems describedabove (individually and/or collectively referred to herein sometimes as“gene delivery vehicles”), in combination with a pharmaceuticallyacceptable carrier or diluent. Such gene delivery vehicles can beformulated in crude or, preferably, purified form. Pharmaceuticalcompositions comprising the gene delivery vehicles may be preparedeither as a liquid solution or as a solid form (e.g., lyophilized) whichis resuspended in a solution prior to administration. In addition, thecomposition may be prepared with suitable carriers or diluents fortopical administration, injection, or nasal, oral, vaginal, sub-lingual,inhalant, intraocular, enteric, or rectal administration.

Pharmaceutically acceptable carrier or diluents are nontoxic torecipients at the dosages and concentrations employed. Representativeexamples of carriers or diluents for injectable solutions include water,isotonic saline solutions, preferably buffered at a physiological pH(such as phosphate-buffered saline or Tris-buffered saline), mannitol,dextrose, glycerol, and ethanol, as well as polypeptides or proteinssuch as human serum albumin (HSA).

Gene delivery vehicles according to the invention can be stored inliquid, or preferably, lyophilized form. Factors influencing stabilityinclude the formulation (liquid, freeze dried, constituents thereof,etc.) and storage conditions, including temperature, storage container,exposure to light, etc. Alternatively, pharmaceutical compositionsaccording to the invention can be stored as liquids at low temperatures.In a preferred embodiment, the gene delivery vehicles of the inventionare formulated to preserve infectivity in a lyophilized form at elevatedtemperatures, and for this form to be suitable for injection intopatients following reconstitution.

In another aspect of the present invention, methods are provided forpreventing or treating various diseases and genetic disorders. Suchmethods comprise administering a gene delivery vehicle as describedabove, such that a therapeutically efficacious amount of the desired, or“selected,” gene product is produced. As used herein, a “therapeuticallyeffective amount” is an amount that that is of clinical relevance, i.e.,protective immunity is achieved, tumor progression is retarded, etc. A“therapeutically effective amount” of a gene delivery vehicle accordingto the invention refers to the amount that must be administered toproduce a therapeutically effective amount of the desired gene productin a particular patient or application. For instance, in a patientsuffering from hemophilia A, a therapeutically effective amount of agene delivery vehicle is an amount that elicits production of sufficientfactor VIII (the desired product expressed from the selectedheterologous nucleotide sequence) to produce therapeutically beneficialclotting and will thus generally be determined by each patient'sattending physician, although serum levels of about 0.2 ng/mL (about0.1% of “normal” levels) or more will be therapeutically beneficial.Typical dosages will range from about 10⁵ to 10¹² gene deliveryvehicles.

In some cases, gene delivery vehicles according to the invention will beadministered as an adjunct to other therapy, such as hormonal,radiation, and/or chemotherapeutic treatment.

In various embodiments of the invention, gene delivery vehicles may beadministered by various routes in vivo, or ex vivo, as described ingreater detail below. Alternatively, the gene delivery vehicles of thepresent invention may also be administered to a patient by a variety ofother methods. Representative examples include transfection by variousphysical methods, such as lipofection (Felgner, et al., Proc. Natl.Acad. Sci. U.S.A., 84:7413: 1989), direct DNA injection (Acsai, et al.,Nature, 352:815, 1991; microprojectile bombardment (Williams, et al.,Proc. Nat'l. Acad. Sci. U.S.A., 88:2726, 1991); liposomes of severaltypes (see e.g., Wang, et al., Proc. Nat'l. Acad. Sci. U.S.A., 84:7851,1987); CaPO₄ (Dubensky, et al., Proc. Nat'l. Acad. Sci. U.S.A. 81:7529,1984); DNA ligand (Wu, et al., J. Biol. Chem., 264:16985, 1989); oradministration of nucleic acids alone (WO 90/11092). Other possiblemethods of administration can include injection of producer cell linesinto the blood or, alternatively, into one or more particular tissues,grafting tissue comprising cells treated with gene delivery vehiclesaccording to the invention, etc.

When pharmaceutical compositions according to the invention areadministered in vivo, i.e., to the cells of patient without priorremoval of the cells from the patient, administration can be by one ormore routes. In this context, “administration” is equivalent to“delivery”. Typical routes of administration include traditionalparenteral routes, such as intramuscular (i.m.), subcutaneous (sub-q),intravenous (i.v.), and interperitoneal (i.p.) injection. Other suitableroutes include nasal, pulmonary, and even direct administration into aparticular tissue, such as the liver, bone marrow, etc. In addition,other routes may be employed, as described below.

Transdermal or topical application of a pharmaceutical compositioncomprising a gene delivery vehicle according to the invention may beused as an alternate route of administration because the skin is themost expansive and readily accessible organ of the human body.Transdermal delivery systems (TDS) are capable of delivering a genedelivery vehicle through intact skin so that it reaches the systemiccirculation in sufficient quantity to be therapeutically effective. TDSprovide a variety of advantages, including elimination ofgastrointestinal absorption problems and hepatic first pass effect,reduction of dosage and dose intervals, and improved patient compliance.The major components of TDS are a controlled release device composed ofpolymers, a gene delivery vehicle according to the invention,excipients, and enhancers, and a fastening system to fix the device tothe skin. A number of polymers have been described and include, but arenot limited to, gelatin, gum arabic, paraffin waxes, and celluloseacetate phthalate (Sogibayashi, et al., J. Controlled Release, 29:177,1994). These polymers can be dermatologically formulated into aqueous,powder, or oil phases. Various combination can produce lotions, pastes,ointments, creams, and gels, alone or together with the aid ofemulsifiers.

Additionally, iontophoresis may be used to cause increased penetrationof ionized substances into or through the skin by the application of anelectrical field. This method has the advantage of being able to deliverthe drug in a pulsatile manner (Singh, et al, Dermatology, 187:235,1993).

Topical administration may also be accomplished be encapsulating genedelivery vehicles according to the invention in liposomes. Hyaluronicacid has been used as a bioadhesive ligand for the formulation ofliposomes to enhance adherence and retention to the extracellular matrixin cases of burns and wound healing (Yerushalmi, et al., Arch. Biochem.and Biophys, 313:267, 1994). As those in the art will appreciate,methods of liposome preparation can be tailored to control size andmorphology. Liposomes can also be made to include one or more targetingelements to target a specific cell type.

Ocular administration is an alternate route to achieve delivery ofcompositions described herein. Systemic absorption occurs throughcontact with the conjunctival and nasal mucosae, the latter occurring asthe result of drainage through the nasolacrimal duct. Formulations suchas those described above which further comprise inert ingredients suchas buffers, chelating agents, antioxidants, and preservatives can beincorporated into ophthalmic dosage forms intended for multiple doseuse. Formulations also may consist of aqueous suspensions, ointments,gels, inserts, bioadhesives, microparticles, and nanoparticles.

The nasal cavity also offers an alternative route of administration forcompositions comprising a gene delivery vehicle as described herein. Forinstance, the human nasal cavities have a total surface area ofapproximately 150 cm² and are covered by a highly vascular mucosallayer. A respiratory epithelium, comprised of columnar cells, gobletcells, and ciliary cuboidal cells, lines most of the nasal cavity(Chien, et al, Crit. Rev. in Therap. Drug Car. Sys., 4:67, 1987). Thesubepithelium contains a dense vascular network and the venous bloodfrom the nose passes directly into the systemic circulation, avoidingfirst-pass metabolism in the liver. Thus, delivery to the upper regionof the nasal cavity may result in slower clearance and increasedbioavailability of gene delivery vehicles. The absence of cilia in thisarea is an important factor in the increased effectiveness of nasalsprays as compared to drops. The addition of viscosity-building agents,such as methycellulose, etc. can change the pattern of deposition andclearance of intranasal applications. Additionally, bioadhesives can beused as a means to prolong residence time in the nasal cavity. Variousformulations comprising sprays, drops, and powders, with or without theaddition of absorptive enhancers, have been described (see Wearley, L,supra).

Oral administration includes sublingual, buccal, and gastrointestinaldelivery. Sublingual and buccal (cheek) delivery allow for rapidsystemic absorption of gene delivery vehicles and avoid hepaticfirst-pass metabolism and degradation in the stomach and intestines.Unidirectional buccal delivery devices can be designed for oral mucosalabsorption only. Additionally, these devices can preventdiffusion-limiting mucus buildup to allow for enhanced absorption.Delivery through the gastrointestinal tract allows for precise targetingfor drug release. Depending on the formulation, gene delivery vehiclescan be specifically delivered to areas in the stomach, duodenum,jejunum, ileum, cecum, colon, or rectum. Oral formulations includetablets, capsules, aqueous suspensions, and gels. These may containbioadhesive polymers, hydrogynamically balanced systems,gastroinflatable delivery devices, intragastric retention shapes,enteric coatings, excipients, or intestinal absorption promoters(Ritschel, W. A., Meth. Exp. Clin. Pharmacol., 13::313, 1991).

The human rectum has a surface area of between 200 to 400 cm² and isabundant in blood and lymphatic vessels. This offers an alternativeroute for administering compositions according to the invention.Depending on the actual site of administration, it may be possible tobypass first-pass metabolism by the liver. Targeting of the systemiccirculation can be achieved by delivering the vehicle to an area behindthe internal rectal sphincter which allows absorption directly into theinferior vena cava, thereby bypassing the portal circulation andavoiding metabolism in the liver. The liver can be targeted bydelivering the vehicle to the region of the ampulla recti, which allowsabsorption into the portal system (Ritschel, supra.).

Alternatively, pulmonary administration can be accomplished throughaerosolization. As the lungs are highly vascularized, this type ofadministration allows systemic delivery. The three systems commonly usedfor aerosol production are: the nebulizer, the pressurized metered doseinhaler, and the dry powder inhaler, all of which are known in the art.Aerosol therapy is very common in obstructive bronchial diseases but canbe used as well as for the treatment of systemic diseases. The surfacearea of the adult human lung is approximately 75 m² and requires onlyone puff of an aerosol to cover this entire area within seconds.Absorption occurs quickly because the walls of the alveoli in the deeplung are extremely thin. Absorption and clearance depends on a number offactors, including particle size and solubility (Wearley, L, supra.).Particles are preferably smaller than 5 μm in diameter.

The vaginal mucosa consists of stratified squamous epithelium. Genedelivery vehicles can be administered through the vaginal orifice ontothe mucosa. Formulations include ointments, creams, and suppositories.Additional information regarding these and other routes ofadministration may be found in U.S. Ser. No. 08/366,788, filed on Dec.20, 1994.

As an alternative to in vivo administration of the gene deliveryvehicles of the invention, ex vivo administration can be employed. Exvivo treatment envisions withdrawal or removal of a population of cellsfrom a patient. Exemplary cell populations include bone marrow cells,liver cells, and blood cells from the umbilical cord of a newborn. Suchcells may be be processed to purify desired cells for transduction priorto such procedures, for instance to obtain subsets of such cellpopulations, e.g., CD34⁺ bone marrow progenitor cells. Preferred methodsof purification include various cell sorting techniques, such asantibody panning, FACS, and affinity chromatography using a matrixcoupled to antibodies specifically reactive to the desired cell type(s).Isolated cells are then transduced, after which they may be immediatelyre-introduced to the patient from which they were withdrawn.Alternatively, the cells may be expanded in culture by varioustechniques known to those skilled in the art prior to re-introduction.

In another embodiment of the invention, gene delivery vehicles of theinvention are administered to patients in conjunction with anothertherapeutic compound. As those in the art will appreciate, suchcompounds may include, but are not limited to, other gene deliveryvehicles designed to delivery one or more other therapeutic genes to thepatient, as is described in U.S. Ser. No. 08/368,210.

In accordance with the non-parenteral administration the presentinvention, the gene delivery vehicles, particularly those comprised ofunencapsidated nucleic acid, may be complexed with a polycationicmolecule to provide polycation-assisted non-parenteral administration.Such a method of gene delivery facilitates delivery of a gene viamediation by a physical particle comprised of multiple components thataugment the efficiency and specificity of the gene transfer. Inparticular, polycationic molecules, such as polylysine and histone, havebeen shown to neutralize the negative charges on a nucleic acid moleculeand to condense the molecule into a compact form. This form of moleculeis transferred with high efficiency in cells, apparently through theendocytic pathway. The uptake in expression of the nucleic acid moleculein the host cell results after a series of steps, as follows: (1)attachment to cell surface; (2) cell entry via endocytosis or othermechanisms; (3) cytoplasmic compartment entry following endosomerelease; (4) nuclear transport; and (5) expression of the nucleic acidmolecule carried by the gene delivery vehicle. In a further preferredembodiment, multi-layer technologies are applied to thepolycation-nucleic acid molecule complex to facilitate completion of oneor more of these steps. For example, a ligand such asasialoglycoprotein, transferrin, and immunoglobulin may be added to thecomplex to facilitate binding of the cell complex to the cell surface,an endosomal disruption component (e.g., a viral protein, a fusogenicpeptide such as the n-terminus of the influenza virus hemaglutinin or aninactivated virus) is added to facilitate the release of DNA from theendosome, or a nuclear protein (or a peptide containing a nuclearlocalization signal) is added to facilitate the transport of the DNAinto the nucleus. In a further preferred embodiment, the compositioncomprising the complex includes inactivated adenovirus particles(Curiel, D. T., et al., PNAS 88: 8850-8854, 1991; Cristiano, R. J., PNAS90: 2122-2126 1993; Cotten, M., et al., PNAS 89: 6094-6098 1992; Lozier,J. N., Human Gene Therapy 5: 313-322, 1994; Curiel D. T., et al., HumanGene Therapy 3: 147-154, 1992; Plank, C. et al., Bioconjugate Chem. 3:533-539, 1992; Wagner, E. et al., PNAS 88: 4255-4259, 1991). Theassorted components comprising the multi-layer complex may be varied asdesired, so that the specificity of the complex for a given tissue, orthe gene expressed from the gene delivery vehicle, may be varied tobetter suit a particular disease or condition.

As noted above, various methods may be utilized to administer genedelivery vehicles of the present invention, including nucleic acidswhich encode the immunogenic portion(s) discussed above, to warm-bloodedanimals such as humans, directly. Suitable methods include, for example,various physical methods such as direct DNA injection (Acsadi et al.,Nature 352:815-818, 1991), and microprojectile bombardment (Williams etal., PNAS 88:2726-2730, 1991).

Within an in vivo context, the gene delivery vehicle can be injectedinto the interstitial space of tissues including muscle, brain, liver,skin, spleen or blood (see, WO 90/11092). Administration may also beaccomplished by intravenous injection or direct catheter infusion intothe cavities of the body (see, WO 93/00051), discussed in more detailbelow.

It is generally preferred that administration of the gene deliveryvehicles at multiple sites be via at least two injections. In thisregard, suitable modes of administration include intramuscular,intradermal and subcutaneous injections, with at least one of theinjections preferably being intramuscular. In particularly preferredembodiments, two or more of the injections are intramuscular. However,although administration via injections is preferred, it will be evidentthat the gene delivery vehicles may be administered through multipletopical or separate ocular administrations. Further, a number ofadditional routes are suitable for use within the present invention whencombined with one or more of the routes briefly noted above, includingintraperitoneal, intracranial, oral, rectal, nasal, vaginal andsublingual administration. Methods of formulating and administering thegene delivery vehicles at multiple sites through such routes would beevident to those skilled in the art and are described in U.S. Ser. No.08/367,071, filed Dec. 30, 1994, incorporated herein by reference intheir entirety.

C. Liposome Formulation

Several methods may be used in the preparation of liposomes toincorporate gene delivery vehicles of the invention, particularly thosethat are DNA or RNA, see Gregoriadis et. al., (Lipsome Technology, CFCPress, New York 1984), Ostro et. al., (Lipsomes, Marek Dekker, 1987) andLichtenberg et. al., (Meth. Biochem. Anal. 33:337, 1988). According toone embodiment of the invention, the gene delivery vehicles arecomplexed with cationic liposomes or lipid vesicles. Cationic liposomeformulations may be prepared from a mixture of positively chargedlipids, negatively charged lipids, neutral lipids and cholesterol orsimilar sterol. The positively charged lipids may be DMRIE (Felgner, et.al., J. Biol. Chem. 269:1, 1994), DOTMA, DOTAP or analogs thereof or acombination of two or more of these lipids. DMRIE is described in U.S.Ser. No. 07/686,746 which is hereby incorporated reference. The neutraland negatively charged lipids can be any natural or syntheticphospholipid or mono-, di- or triglycerols. The natural phospholipidsmay be derived from animal and plant sources. For example, naturalphospholipids such as phosphotidylcholine, phosphotidylethanolamine,sphingomylin, phosphotidylserine, or phosphotidylinositol may beutilized. Synthetic phospholipids may be selected from those havingfatty acid groups such as dimyristoylphophatidylcholine,distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine,disteroylphophatidylcholine, and the correspondingphophatidylethanolamines and phosphatidylglycerols. The neutral lipidsmay be phosphatidylcholine, cardiolipin, phosphatidylethanolamine,mono-, di- or triacylglycerols, or analogs thereof such asdioleoylphosphatidylethanolamine (DOPE). The negatively charged lipidsmay be phosphatidylglycerol, phosphatidic acid or a similar phospholipidanalog. Other additive known to those skilled in the art may also beused such as cholesterol, glycolipids, fatty acids, sphingolipids,prostaglandins, gangliosides, neobee, niomes, or any other natural orsynthetic amphophilies.

Substitution of the cationic lipid component of liposomes may be used toalter the transfection efficiency of the liposome. For example,1,2-dimyristyloxypropyl-3-dimethylhydroxyethyl ammonium bromide (DMRIE)is used in conjunction with DOPE which provides increased transfectionefficiency and does not aggregate at high concentrations as otherformulations such as DC-cholesterol/DOPE. These characteristics allowsfor higher absolute concentrations of DNA and liposomes to be introducedinto patients in vivo without increased levels of toxicity. A preferredmolar ratio of DMRIE to DOPE of 9:1 to 1:9 with a particularly preferredmolar ratio of 5:5 (see WO 94/29469 incorporated herein by reference)

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

Additionally, the publications and other materials cited to illuminatethe background of the invention, and in particular, to provideadditional details concerning its practice as described in the detaileddescription and examples, are hereby incorporated by reference in theirentirety.

A Sequence Listing has also been included herewith in accordance withthe provisions of 37 C.F.R. §1.821 et seq. To the extent any discrepancyexists between the Specification Figures and the Sequence Listing, thespecification or Figures should be considered to be the primarydocument.

128 16656 base pairs nucleic acid single linear 1 ATTGACGGCG TAGTACACACTATTGAATCA AACAGCCGAC CAATCGCACT ACCATCACAA 60 TGGAGAAGCC AGTAGTAAACGTAGACGTAG ACCCCCAGAG TCCGTTTGTC GTGCAACTGC 120 AAAAAAGCTT CCCGCAATTTGAGGTAGTAG CACAGCAGGT CACTCCAAAT GACCATGCTA 180 ATGCCAGAGC ATTTTCGCATCTGGCCAGTA AACTAATCGA GCTGGAGGTT CCTACCACAG 240 CGACGATCTT GGACATAGGCAGCGCACCGG CTCGTAGAAT GTTTTCCGAG CACCAGTATC 300 ATTGTGTCTG CCCCATGCGTAGTCCAGAAG ACCCGGACCG CATGATGAAA TATGCCAGTA 360 AACTGGCGGA AAAAGCGTGCAAGATTACAA ACAAGAACTT GCATGAGAAG ATTAAGGATC 420 TCCGGACCGT ACTTGATACGCCGGATGCTG AAACACCATC GCTCTGCTTT CACAACGATG 480 TTACCTGCAA CATGCGTGCCGAATATTCCG TCATGCAGGA CGTGTATATC AACGCTCCCG 540 GAACTATCTA TCATCAGGCTATGAAAGGCG TGCGGACCCT GTACTGGATT GGCTTCGACA 600 CCACCCAGTT CATGTTCTCGGCTATGGCAG GTTCGTACCC TGCGTACAAC ACCAACTGGG 660 CCGACGAGAA AGTCCTTGAAGCGCGTAACA TCGGACTTTG CAGCACAAAG CTGAGTGAAG 720 GTAGGACAGG AAAATTGTCGATAATGAGGA AGAAGGAGTT GAAGCCCGGG TCGCGGGTTT 780 ATTTCTCCGT AGGATCGACACTTTATCCAG AACACAGAGC CAGCTTGCAG AGCTGGCATC 840 TTCCATCGGT GTTCCACTTGAATGGAAAGC AGTCGTACAC TTGCCGCTGT GATACAGTGG 900 TGAGTTGCGA AGGCTACGTAGTGAAGAAAA TCACCATCAG TCCCGGGATC ACGGGAGAAA 960 CCGTGGGATA CGCGGTTACACACAATAGCG AGGGCTTCTT GCTATGCAAA GTTACTGACA 1020 CAGTAAAAGG AGAACGGGTATCGTTCCCTG TGTGCACGTA CATCCCGGCC ACCATATGCG 1080 ATCAGATGAC TGGTCTAATGGCCACGGATA TATCACCTGA CGATGCACAA AAACTTCTGG 1140 TTGGGCTCAA CCAGCGAATTGTCATTAACG GTAGGACTAA CAGGAACACC AACACCATGC 1200 AAAATTACCT TCTGCCGATCATAGCACAAG GGTTCAGCAA ATGGGCTAAG GAGCGCAAGG 1260 ATGATCTTGA TAACGAGAAAATGCTGGGTA CTAGAGAACG CAAGCTTACG TATGGCTGCT 1320 TGTGGGCGTT TCGCACTAAGAAAGTACATT CGTTTTATCG CCCACCTGGA ACGCAGACCT 1380 GCGTAAAAGT CCCAGCCTCTTTTAGCGCTT TCCCCATGTC GTCCGTATGG ACGACCTCTT 1440 TGCCCATGTC GCTGAGGCAGAAATTGAAAC TGGCATTGCA ACCAAAGAAG GAGGAAAAAC 1500 TGCTGCAGGT CTCGGAGGAATTAGTCATGG AGGCCAAGGC TGCTTTTGAG GATGCTCAGG 1560 AGGAAGCCAG AGCGGAGAAGCTCCGAGAAG CACTTCCACC ATTAGTGGCA GACAAAGGCA 1620 TCGAGGCAGC CGCAGAAGTTGTCTGCGAAG TGGAGGGGCT CCAGGCGGAC ATCGGAGCAG 1680 CATTAGTTGA AACCCCGCGCGGTCACGTAA GGATAATACC TCAAGCAAAT GACCGTATGA 1740 TCGGACAGTA TATCGTTGTCTCGCCAAACT CTGTGCTGAA GAATGCCAAA CTCGCACCCG 1800 CGCACCCGCT AGCAGATCAGGTTAAGATCA TAACACACTC CGGAAGATCA GGAAGGTACG 1860 CGGTCGAACC ATACGACGCTAAAGTACTGA TGCCAGCAGG AGGTGCCGTA CCATGGCCAG 1920 AATTCCTAGC ACTGAGTGAGAGCGCCACGT TAGTGTACAA CGAAAGAGAG TTTGTGAACC 1980 GCAAACTATA CCACATTGCCATGCATGGCC CCGCCAAGAA TACAGAAGAG GGGCAGTACA 2040 AGGTTACAAA GGCAGAGCTTGCAGAAACAG AGTACGTGTT TGACGTGGAC AAGAAGCGTT 2100 GCGTTAAGAA GGAAGAAGCCTCAGGTCTGG TCCTCTCGGG AGAACTGACC AACCCTCCCT 2160 ATCATGAGCT AGCTCTGGAGGGACTGAAGA CCCGACCTGC GGTCCCGTAC AAGGTCGAAA 2220 CAATAGGAGT GATAGGCACACCGGGGTCGG GCAAGTCCGC TATTATCAAG TCAACTGTCA 2280 CGGCACGAGA TCTTGTTACCAGCGGAAAGA AAGAAAATTG TCGCGAAATT GAGGCCGACG 2340 TGCTAAGACT GAGGGGTATGCAGATTACGT CGAAGACAGT AGATTCGGTT ATGCTCAACG 2400 GATGCCACAA AGCCGTAGAAGTGCTGTACG TTGACGAAGC GTTCGCGTGC CACGCAGGAG 2460 CACTACTTGC CTTGATTGCTATCGTCAGGC CCCGCAAGAA GGTAGTACTA TGCGGAGACC 2520 CCATGCAATG CGGATTCTTCAACATGATGC AACTAAAGGT ACATTTCAAT CACCCTGAAA 2580 AAGACATATG CACCAAGACATTCTACAAGT ATATCTCCCG GCGTTGCACA CAGCCAGTTA 2640 CAGCTATTGT ATCGACACTGCATTACGATG GAAAGATGAA AACCACGAAC CCGTGCAAGA 2700 AGAACATTGA AATCGATATTACAGGGGCCA CAAAGCCGAA GCCAGGGGAT ATCATCCTGA 2760 CATGTTTCCG CGGGTGGGTTAAGCAATTGC AAATCGACTA TCCCGGACAT GAAGTAATGA 2820 CAGCCGCGGC CTCACAAGGGCTAACCAGAA AAGGAGTGTA TGCCGTCCGG CAGAAAGTCA 2880 ATGAAAACCC ACTGTACGCGATCACATCAG AGCATGTGAA CGTGTTGCTC ACCCGCACTG 2940 AGGACAGGCT AGTGTGGAAAACCTTGCAGG GCGACCCATG GATTAAGCAG CTCACTAACA 3000 TACCTAAAGG AAACTTTCAGGCTACTATAG AGGACTGGGA AGCTGAACAC AAGGGAATAA 3060 TTGCTGCAAT AAACAGCCCCACTCCCCGTG CCAATCCGTT CAGCTGCAAG ACCAACGTTT 3120 GCTGGGCGAA AGCATTGGAACCGATACTAG CCACGGCCGG TATCGTACTT ACCGGTTGCC 3180 AGTGGAGCGA ACTGTTCCCACAGTTTGCGG ATGACAAACC ACATTCGGCC ATTTACGCCT 3240 TAGACGTAAT TTGCATTAAGTTTTTCGGCA TGGACTTGAC AAGCGGACTG TTTTCTAAAC 3300 AGAGCATCCC ACTAACGTACCATCCCGCCG ATTCAGCGAG GCCGGTAGCT CATTGGGACA 3360 ACAGCCCAGG AACCCGCAAGTATGGGTACG ATCACGCCAT TGCCGCCGAA CTCTCCCGTA 3420 GATTTCCGGT GTTCCAGCTAGCTGGGAAGG GCACACAACT TGATTTGCAG ACGGGGAGAA 3480 CCAGAGTTAT CTCTGCACAGCATAACCTGG TCCCGGTGAA CCGCAATCTT CCTCACGCCT 3540 TAGCCCCCGA GTACAAGGAGAAGCAACCCG GCCCGGTCGA AAAATTCTTG AACCAGTTCA 3600 AACACCACTC AGTACTTGTGGTATCAGAGG AAAAAATTGA AGCTCCCCGT AAGAGAATCG 3660 AATGGATCGC CCCGATTGGCATAGCCGGTG CAGATAAGAA CTACAACCTG GCTTTCGGGT 3720 TTCCGCCGCA GGCACGGTACGACCTGGTGT TCATCAACAT TGGAACTAAA TACAGAAACC 3780 ACCACTTTCA GCAGTGCGAAGACCATGCGG CGACCTTAAA AGCCCTTTCG CGTTCGGCCC 3840 TGAATTGCCT CAACCCAGGAGGCACCCTCG TGGTGAAGTC CTATGGCTAC GCCGACCGCA 3900 ACAGTGAGGA CGTAGTCACCGCTCTTGCCA GAAAGTTTGT CAGGGTGTCT GCAGCGAGAC 3960 CAGATTGTGT CTCAAGCAATACAGAAATGT ACCTGATTTT CCGACAACTA GACAACAGCC 4020 GTACACGGCA ATTCACCCCGCACCATCTGA ATTGCGTGAT TTCGTCCGTG TATGAGGGTA 4080 CAAGAGATGG AGTTGGAGCCGCGCCGTCAT ACCGCACCAA AAGGGAGAAT ATTGCTGACT 4140 GTCAAGAGGA AGCAGTTGTCAACGCAGCCA ATCCGCTGGG TAGACCAGGC GAAGGAGTCT 4200 GCCGTGCCAT CTATAAACGTTGGCCGACCA GTTTTACCGA TTCAGCCACG GAGACAGGCA 4260 CCGCAAGAAT GACTGTGTGCCTAGGAAAGA AAGTGATCCA CGCGGTCGGC CCTGATTTCC 4320 GGAAGCACCC AGAAGCAGAAGCCTTGAAAT TGCTACAAAA CGCCTACCAT GCAGTGGCAG 4380 ACTTAGTAAA TGAACATAACATCAAGTCTG TCGCCATTCC ACTGCTATCT ACAGGCATTT 4440 ACGCAGCCGG AAAAGACCGCCTTGAAGTAT CACTTAACTG CTTGACAACC GCGCTAGACA 4500 GAACTGACGC GGACGTAACCATCTATTGCC TGGATAAGAA GTGGAAGGAA AGAATCGACG 4560 CGGCACTCCA ACTTAAGGAGTCTGTAACAG AGCTGAAGGA TGAAGATATG GAGATCGACG 4620 ATGAGTTAGT ATGGATCCATCCAGACAGTT GCTTGAAGGG AAGAAAGGGA TTCAGTACTA 4680 CAAAAGGAAA ATTGTATTCGTACTTCGAAG GCACCAAATT CCATCAAGCA GCAAAAGACA 4740 TGGCGGAGAT AAAGGTCCTGTTCCCTAATG ACCAGGAAAG TAATGAACAA CTGTGTGCCT 4800 ACATATTGGG TGAGACCATGGAAGCAATCC GCGAAAAGTG CCCGGTCGAC CATAACCCGT 4860 CGTCTAGCCC GCCCAAAACGTTGCCGTGCC TTTGCATGTA TGCCATGACG CCAGAAAGGG 4920 TCCACAGACT TAGAAGCAATAACGTCAAAG AAGTTACAGT ATGCTCCTCC ACCCCCCTTC 4980 CTAAGCACAA AATTAAGAATGTTCAGAAGG TTCAGTGCAC GAAAGTAGTC CTGTTTAATC 5040 CGCACACTCC CGCATTCGTTCCCGCCCGTA AGTACATAGA AGTGCCAGAA CAGCCTACCG 5100 CTCCTCCTGC ACAGGCCGAGGAGGCCCCCG AAGTTGTAGC GACACCGTCA CCATCTACAG 5160 CTGATAACAC CTCGCTTGATGTCACAGACA TCTCACTGGA TATGGATGAC AGTAGCGAAG 5220 GCTCACTTTT TTCGAGCTTTAGCGGATCGG ACAACTCTAT TACTAGTATG GACAGTTGGT 5280 CGTCAGGACC TAGTTCACTAGAGATAGTAG ACCGAAGGCA GGTGGTGGTG GCTGACGTTC 5340 ATGCCGTCCA TGAGCCTGCCCCTATTCCAC CGCCAAGGCT AAAGAAGATG GCCCGCCTGG 5400 CAGCGGCAAG AAAAGAGCCCACTCCACCGG CAAGCAATAG CTCTGAGTCC CTCCACCTCT 5460 CTTTTGGTGG GGTATCCATGTCCCTCGGAT CAATTTTCGA CGGAGAGACG GCCCGCCAGG 5520 CAGCGGTACA ACCCCTGGCAACAGGCCCCA CGGATGTGCC TATGTCTTTC GGATCGTTTT 5580 CCGACGGAGA GATTGATGAGCTGAGCCGCA GAGCAACTGA GTCCGAACCC GTCCTGTTTG 5640 GATCATTTGA ACCGGGCGAAGTGAACTCAA TTATATCGTC CCGATCAGCC GTATCTTTTC 5700 CACTACGCAA GCAGAGACGTAGACGCAGGA GCAGGAGGAC TGAATACTGA CTAACCGGGG 5760 TAGGTGGGTA CATATTTTCGACGGACACAG GCCCTGGGCA CTTGCAAAAG AAGTCCGTTC 5820 TGCAGAACCA GCTTACAGAACCGACCTTGG AGCGCAATGT CCTGGAAAGA ATTCATGCCC 5880 CGGTGCTCGA CACGTCGAAAGAGGAACAAC TCAAACTCAG GTACCAGATG ATGCCCACCG 5940 AAGCCAACAA AAGTAGGTACCAGTCTCGTA AAGTAGAAAA TCAGAAAGCC ATAACCACTG 6000 AGCGACTACT GTCAGGACTACGACTGTATA ACTCTGCCAC AGATCAGCCA GAATGCTATA 6060 AGATCACCTA TCCGAAACCATTGTACTCCA GTAGCGTACC GGCGAACTAC TCCGATCCAC 6120 AGTTCGCTGT AGCTGTCTGTAACAACTATC TGCATGAGAA CTATCCGACA GTAGCATCTT 6180 ATCAGATTAC TGACGAGTACGATGCTTACT TGGATATGGT AGACGGGACA GTCGCCTGCC 6240 TGGATACTGC AACCTTCTGCCCCGCTAAGC TTAGAAGTTA CCCGAAAAAA CATGAGTATA 6300 GAGCCCCGAA TATCCGCAGTGCGGTTCCAT CAGCGATGCA GAACACGCTA CAAAATGTGC 6360 TCATTGCCGC AACTAAAAGAAATTGCAACG TCACGCAGAT GCGTGAACTG CCAACACTGG 6420 ACTCAGCGAC ATTCAATGTCGAATGCTTTC GAAAATATGC ATGTAATGAC GAGTATTGGG 6480 AGGAGTTCGC TCGGAAGCCAATTAGGATTA CCACTGAGTT TGTCACCGCA TATGTAGCTA 6540 GACTGAAAGG CCCTAAGGCCGCCACACTAT TTGCAAAGAC GTATAATTTG GTCCCATTGC 6600 AAGAAGTGCC TATGGATAGATTCGTCATGG ACATGAAAAG AGACGTGAAA GTTACACCAG 6660 GCACGAAACA CACAGAAGAAAGACCGAAAG TACAAGTGAT ACAAGCCGCA GAACCCCTGG 6720 CGACTGCTTA CTTATGCGGGATTCACCGGG AATTAGTGCG TAGGCTTACG GCCGTCTTGC 6780 TTCCAAACAT TCACACGCTTTTTGACATGT CGGCGGAGGA TTTTGATGCA ATCATAGCAG 6840 AACACTTCAA GCAAGGCGACCCGGTACTGG AGACGGATAT CGCATCATTC GACAAAAGCC 6900 AAGACGACGC TATGGCGTTAACCGGTCTGA TGATCTTGGA GGACCTGGGT GTGGATCAAC 6960 CACTACTCGA CTTGATCGAGTGCGCCTTTG GAGAAATATC ATCCACCCAT CTACCTACGG 7020 GTACTCGTTT TAAATTCGGGGCGATGATGA AATCCGGAAT GTTCCTCACA CTTTTTGTCA 7080 ACACAGTTTT GAATGTCGTTATCGCCAGCA GAGTACTAGA AGAGCGGCTT AAAACGTCCA 7140 GATGTGCAGC GTTCATTGGCGACGACAACA TCATACATGG AGTAGTATCT GACAAAGAAA 7200 TGGCTGAGAG GTGCGCCACCTGGCTCAACA TGGAGGTTAA GATCATCGAC GCAGTCATCG 7260 GTGAGAGACC ACCTTACTTCTGCGGCGGAT TTATCTTGCA AGATTCGGTT ACTTCCACAG 7320 CGTGCCGCGT GGCGGATCCCCTGAAAAGGC TGTTTAAGTT GGGTAAACCG CTCCCAGCCG 7380 ACGACGAGCA AGACGAAGACAGAAGACGCG CTCTGCTAGA TGAAACAAAG GCGTGGTTTA 7440 GAGTAGGTAT AACAGGCACTTTAGCAGTGG CCGTGACGAC CCGGTATGAG GTAGACAATA 7500 TTACACCTGT CCTACTGGCATTGAGAACTT TTGCCCAGAG CAAAAGAGCA TTCCAAGCCA 7560 TCAGAGGGGA AATAAAGCATCTCTACGGTG GTCCTAAATA GTCAGCATAG TACATTTCAT 7620 CTGACTAATA CTACAACACCACCACCATGA ATAGAGGATT CTTTAACATG CTCGGCCGCC 7680 GCCCCTTCCC GGCCCCCACTGCCATGTGGA GGCCGCGGAG AAGGAGGCAG GCGGCCCCGA 7740 TGCCTGCCCG CAACGGGCTGGCTTCTCAAA TCCAGCAACT GACCACAGCC GTCAGTGCCC 7800 TAGTCATTGG ACAGGCAACTAGACCTCAAC CCCCACGTCC ACGCCCGCCA CCGCGCCAGA 7860 AGAAGCAGGC GCCCAAGCAACCACCGAAGC CGAAGAAACC AAAAACGCAG GAGAAGAAGA 7920 AGAAGCAACC TGCAAAACCCAAACCCGGAA AGAGACAGCG CATGGCACTT AAGTTGGAGG 7980 CCGACAGATT GTTCGACGTCAAGAACGAGG ACGGAGATGT CATCGGGCAC GCACTGGCCA 8040 TGGAAGGAAA GGTAATGAAACCTCTGCACG TGAAAGGAAC CATCGACCAC CCTGTGCTAT 8100 CAAAGCTCAA ATTTACCAAGTCGTCAGCAT ACGACATGGA GTTCGCACAG TTGCCAGTCA 8160 ACATGAGAAG TGAGGCATTCACCTACACCA GTGAACACCC CGAAGGATTC TATAACTGGC 8220 ACCACGGAGC GGTGCAGTATAGTGGAGGTA GATTTACCAT CCCTCGCGGA GTAGGAGGCA 8280 GAGGAGACAG CGGTCGTCCGATCATGGATA ACTCCGGTCG GGTTGTCGCG ATAGTCCTCG 8340 GTGGCGCTGA TGAAGGAACACGAACTGCCC TTTCGGTCGT CACCTGGAAT AGTAAAGGGA 8400 AGACAATTAA GACGACCCCGGAAGGGACAG AAGAGTGGTC CGCAGCACCA CTGGTCACGG 8460 CAATGTGTTT GCTCGGAAATGTGAGCTTCC CATGCGACCG CCCGCCCACA TGCTATACCC 8520 GCGAACCTTC CAGAGCCCTCGACATCCTTG AAGAGAACGT GAACCATGAG GCCTACGATA 8580 CCCTGCTCAA TGCCATATTGCGGTGCGGAT CGTCTGGCAG AAGCAAAAGA AGCGTCGTTG 8640 ACGACTTTAC CCTGACCAGCCCCTACTTGG GCACATGCTC GTACTGCCAC CATACTGAAC 8700 CGTGCTTCAG CCCTGTTAAGATCGAGCAGG TCTGGGACGA AGCGGACGAT AACACCATAC 8760 GCATACAGAC TTCCGCCCAGTTTGGATACG ACCAAAGCGG AGCAGCAAGC GCAAACAAGT 8820 ACCGCTACAT GTCGCTTAAGCAGGATCACA CCGTTAAAGA AGGCACCATG GATGACATCA 8880 AGATTAGCAC CTCAGGACCGTGTAGAAGGC TTAGCTACAA AGGATACTTT CTCCTCGCAA 8940 AATGCCCTCC AGGGGACAGCGTAACGGTTA GCATAGTGAG TAGCAACTCA GCAACGTCAT 9000 GTACACTGGC CCGCAAGATAAAACCAAAAT TCGTGGGACG GGAAAAATAT GATCTACCTC 9060 CCGTTCACGG TAAAAGAATTCCTTGCACAG TGTACGACCG TCTGAAAACA ACTGCAGGCT 9120 ACATCACTAT GCACAGGCCGGGACCGCACG CTTATACATC CTACCTGGAA GAATCATCAG 9180 GGAAAGTTTA CGCAAAGCCGCCATCTGGGA AGAACATTAC GTATGAGTGC AAGTGCGGCG 9240 ACTACAAGAC CGGAACCGTTTCGACCCGCA CCGAAATCAC TGGTTGCACC GCCATCAAGC 9300 AGTGCGTCGC CTATAAGAGCGACCAAACGA AGTGGGTCTT CAACTCACCG GACTTGATCA 9360 GACATGACGA CCACACGGCCCAAGGGAAAT TGCATTTGCC TTTCAAGTTG ATCCCGGGTG 9420 CCTGCATGGT CCCTGTTGCCCACGCGCCGA ATGTAATACA TGGCTTTAAA CACATCAGCC 9480 TCCAATTAGA TACAGACCACTTGACATTGC TCACCACCAG GAGACTAGGG GCAAACCCGG 9540 AACCAACCAC TGAATGGATCGTCGGAAAGA CGGTCAGAAA CTTCACCGTC GACCGAGATG 9600 GCCTGGAATA CATATGGGGAAATCATGAGC CAGTGAGGGT CTATGCCCAA GAGTCAGCAC 9660 CAGGAGACCC TCACGGATGGCCACACGAAA TAGTACAGCA TTACTACCAT CGCCATCCTG 9720 TGTACACCAT CTTAGCCGTCGCATCAGCTA CCGTGGCGAT GATGATTGGC GTAACTGTTG 9780 CAGTGTTATG TGCCTGTAAAGCGCGCCGTG AGTGCCTGAC GCCATACGCC CTGGCCCCAA 9840 ACGCCGTAAT CCCAACTTCGCTGGCACTCT TGTGCTGCGT TAGGTCGGCC AATGCTGAAA 9900 CGTTCACCGA GACCATGAGTTACTTGTGGT CGAACAGTCA GCCGTTCTTC TGGGTCCAGT 9960 TGTGCATACC TTTGGCCGCGTTCATCGTTC TAATGCGCTA CTGCTCCTGC TGCCTGCCTT 10020 TTTTAGTGGT TGCCGGCGCCTACCTGGCGA AGGTAGACGC CTACGAACAT GCGACCACTG 10080 TTCCAAATGT GCCACAGATACCGTATAAGG CACTTGTTGA AAGGGCAGGG TATGCCCCCG 10140 TCAATTTGGA GATCACTGTCATGTCCTCGG AGGTTTTGCC TTCCACCAAC CAAGAGTAGA 10200 TTACCTGCAA ATTCACCACTGTGGTCCCCT CCCCAAAAAT CAAATGCTGC GGCTCCTTGG 10260 AATGTCAGCC GGCCGCTCATGCAGACTATA CCTGCAAGGT CTTCGGAGGG GTCTACCCTT 10320 TTATGTGGGG AGGAGCGCAATGTTTTTGCG ACAGTGAGAA CAGCCAGATG AGTGAGGCGT 10380 ACGTCGAATT GTCAGCAGATTGCGCGTCTG ACCACGCGCA GGCGATTAAG GTGCACACTG 10440 CCGCGATGAA AGTAGGACTGCGTATAGTGT ACGGGAACAC TACCAGTTTC CTAGATGTGT 10500 ACGTGAACGG AGTCACACCAGGAACGTCTA AAGACTTGAA AGTCATAGCT GGACCAATTT 10560 CAGCATCGTT TACGCCATTCGATCATAAGG TCGTTATCCA TCGCGGCCTG GTGTACAACT 10620 ATGACTTCCC GGAATATGGAGCGATGAAAC CAGGAGCGTT CGGAGACATT CAAGCTACCT 10680 CCTTGACTAG CAAGGATCTCATCGCCAGCA CAGACATTAG GCTACTCAAG CCTTCCGCCA 10740 AGAACGTGCA TGTCCCGTACACGCAGGCCG CATCAGGATT TGAGATGTGG AAAAACAACT 10800 CAGGCCGCCC ACTGCAGGAAACCGCACCTT TCGGGTGTAA GATTGCAGTA AATCCGCTCC 10860 GAGCGGTGGA CTGTTCATACGGGAACATTC CCATTTCTAT TGACATCCCG AACGCTGCCT 10920 TTATCAGGAC ATCAGATGCACCACTGGTCT CAACAGTCAA ATGTGAAGTC AGTGAGTGCA 10980 CTTATTCAGC AGACTTCGGCGGGATGGCCA CCCTGCAGTA TGTATCCGAC CGCGAAGGTC 11040 AATGCCCCGT ACATTCGCATTCGAGCACAG CAACTCTCCA AGAGTCGACA GTACATGTCC 11100 TGGAGAAAGG AGCGGTGACAGTACACTTTA GCACCGCGAG TCCACAGGCG AACTTTATCG 11160 TATCGCTGTG TGGGAAGAAGACAACATGCA ATGCAGAATG TAAACCACCA GCTGACCATA 11220 TCGTGAGCAC CCCGCACAAAAATGACCAAG AATTTCAAGC CGCCATCTCA AAAACATCAT 11280 GGAGTTGGCT GTTTGCCCTTTTCGGCGGCG CCTCGTCGCT ATTAATTATA GGACTTATGA 11340 TTTTTGCTTG CAGCATGATGCTGACTAGCA CACGAAGATG ACCGCTACGC CCCAATGATC 11400 CGACCAGCAA AACTCGATGTACTTCCGAGG AACTGATGTG CATAATGCAT CAGGCTGGTA 11460 CATTAGATCC CCGCTTACCGCGGGCAATAT AGCAACACTA AAAACTCGAT GTACTTCCGA 11520 GGAAGCGCAG TGCATAATGCTGCGCAGTGT TGCCACATAA CCACTATATT AACCATTTAT 11580 CTAGCGGACG CCAAAAACTCAATGTATTTC TGAGGAAGCG TGGTGCATAA TGCCACGCAG 11640 CGTCTGCATA ACTTTTATTATTTCTTTTAT TAATCAACAA AATTTTGTTT TTAACATTTC 11700 AAAAAAAAAA AAAAAAAAAAAAAAATCTAG AGGGCCCTAT TCTATAGTGT CACCTAAATG 11760 CTAGAGCTCG CTGATCAGCCTCGACTGTGC CTTCTAGTTG CCAGCCATCT GTTGTTTGCC 11820 CCTCCCCCGT GCCTTCCTTGACCCTGGAAG GTGCCACTCC CACTGTCCTT TCCTAATAAA 11880 ATGAGGAAAT TGCATCGCATTGTCTGAGTA GGTGTCATTC TATTCTGGGG GGTGGGGTGG 11940 GGCAGGACAG CAAGGGGGAGGATTGGGAAG ACAATAGCAG GCATGCTGGG GATGCGGTGG 12000 GCTCTATGGC TTCTGAGGCGGAAAGAACCA GCTGGGGCTC TAGGGGGTAT CCCCACGCGC 12060 CCTGTAGCGG CGCATTAAGCGCGGCGGGTG TGGTGGTTAC GCGCAGCGTG ACCGCTAACA 12120 TTGCCAGCGC CCTAGCGCCCGCTCCTTTCG CTTTCTTCCC TTCCTTTCTC GCCACGTTCG 12180 CCGGCTTTCC CCGTCAAGCTCTAAATCGGG GCATCCCTTT AGGGTTCCGA TTTAGTGCTT 12240 TACGGCACCT CGACCCCAAAAAACTTGATT AGGGTGATGG TTCACGTAGT GGGCCATCGC 12300 CCTGATAGAC GGTTTTTCGCCCTTTGACGT TGGAGTCCAC GTTCTTTAAT AGTGGACTCT 12360 TGTTCCAAAC TGGAACAACACTCAACCCTA TCTCGGTCTA TTCTTTTGAT TTATAAGGAA 12420 TTTTGGGGAT TTCGGCCTATTGGTTAAAAA ATGAGCTGAT TTAACAAAAA TTTAACGCGA 12480 ATTAATTCTG TGGAATGTGTGTCAGTTAGG GTGTGGAAAG TCCCCAGGCT CCCCAGGCAG 12540 GCAGAAGTAT GCAAAGCATGCATCTCAATT AGTCAGCAAC CAGGTGTGGA AAGTCCCCAG 12600 GCTCCCCAGC AGGCAGAAGTATGCAAAGCA TGCATCTCAA TTAGTCAGCA ACCATAGTCC 12660 CGCCCCTAAC TCCGCCCATCCCGCCCCTAA CTCCGCCCAG TTCCGCCCAT TCTCCGCCCC 12720 ATGGCTGACT AATTTTTTTTATTTATGCAG AGGCCGAGGC CGCCTCTGCC TCTGAGCTAT 12780 TCCAGAAGTA GTGAGGAGGCTTTTTTGGAG GCCTAGGCTT TTGCAAAAAG CTCCCGGGAG 12840 CTTGTATATC CATTTTCGGATCTGATCAAG AGACAGGATG AGGATCGTTT CGCATGATTG 12900 AACAAGATGG ATTGCACGCAGGTTCTCCGG CCGCTTGGGT GGAGAGGCTA TTCGGCTATG 12960 ACTGGGCACA ACAGACAATCGGCTGCTCTG ATGCCGCCGT GTTCCGGCTG TCAGCGCAGG 13020 GGCGCCCGGT TCTTTTTGTCAAGACCGACC TGTCCGGTGC CCTGAATGAA CTGCAGGACG 13080 AGGCAGCGCG GCTATCGTGGCTGGCCACGA CGGGCGTTCC TTGCGCAGCT GTGCTCGACG 13140 TTGTCACTGA AGCGGGAAGGGACTGGCTGC TATTGGGCGA AGTGCCGGGG CAGGATCTCC 13200 TGTCATCTCA CCTTGCTCCTGCCGAGAAAG TATCCATCAT GGCTGATGCA ATGCGGCGGC 13260 TGCATACGCT TGATCCGGCTACCTGCCCAT TCGACCACCA AGCGAAACAT CGCATCGTCC 13320 GAGCACGTAC TCGGATGGAAGCCGGTCTTG TCGATCAGGA TGATCTGGAC GAAGAGCACG 13380 AGGGGCTCGC GCCAGCCGAACTGTTCGCCA GGCTCAAGGC GCGCATGCCC GACGGCGAGG 13440 ATCTCGTCGT GACCCATGGCGATGCCTGCT TGCCGAATAT CATGGTGGAA AATGGCCGCT 13500 TTTCTGGATT CATCGACTGTGGCCGGCTGG GTGTGGCGGA CCGCTATCAG GACATAGCGT 13560 TGGCTACCCG TGATATTGCTGAAGAGCTTG GCGGCGAATG GGCTGACCGC TTCCTCGTCG 13620 TTTACGGTAT CGCCGCTCCCGATTCGCAGC GCATCGCCTT CTATCGCCTT CTTGACGAGT 13680 TCTTCTGAGC GGGACTCTGGGGTTCGAAAT GACCGACCAA GCGACGCCCA ACCTGCCATC 13740 ACGAGATTTC GATTCCACCGCCGCCTTCTA TGAAAGGTTG GGCTTCGGAA TCGTTTTCCG 13800 GGACGCCGGC TGGATGATCCTCCAGCGCGG GGATCTCATG CTGGAGTTCT TCGCCCACCC 13860 CAACTTGTTT ATTGCAGCTTATAATGGTTA CAAATAAAGC AATAGCATCA CAAATTTCAC 13920 AAATAAAGCA TTTTTTTCACTGCATTCTAG TTGTGGTTTG TCCAAACTCA TCAATGTATC 13980 TTATCATGTC TGTATACCGTCGACCTCTAG CTAGAGCTTG GCGTAATCAT GGTCATAGCT 14040 GTTTCCTGTG TGAAATTGTTATCCGCTCAC AATTCCACAC AACATACGAG CCGGAAGCAT 14100 AAAGTGTAAA GCCTGGGGTGCCTAATGAGT GAGCTAACTC ACATTAATTG CGTTGCGCTC 14160 ACTGCCCGCT TTCCAGTCGGGAAACCTGTC GTGCCAGCTG CATTAATGAA TCGGCCAACG 14220 CGCGGGGAGA GGCGGTTTGCGTATTGGGCG CTCTTCCGCT TCCTCGCTCA CTGACTCGCT 14280 GCGCTCGGTC GTTCGGCTGCGGCGAGCGGT ATCAGCTCAC TCAAAGGCGG TAATACGGTT 14340 ATCCACAGAA TCAGGGGATAACGCAGGAAA GAACATGTGA GCAAAAGGCC AGCAAAAGGC 14400 CAGGAACCGT AAAAAGGCCGCGTTGCTGGC GTTTTTCCAT AGGCTCCGCC CCCCTGACGA 14460 GCATCACAAA AATCGACGCTCAAGTCAGAG GTGGCGAAAC CCGACAGGAC TATAAAGATA 14520 CCAGGCGTTT CCCCCTGGAAGCTCCCTCGT GCGCTCTCCT GTTCCGACCC TGCCGCTTAC 14580 CGGATACCTG TCCGCCTTTCTCCCTTCGGG AAGCGTGGCG CTTTCTCAAT GCTCACGCTC 14640 TAGGTATCTC AGTTCGGTGTAGGTCGTTCG CTCCAAGCTG GGCTGTGTGC ACGAACCCCC 14700 CGTTCAGCCC GACCGCTGCGCCTTATCCGG TAACTATCGT CTTGAGTCCA ACCCGGTAAG 14760 ACACGACTTA TCGCCACTGGCAGCAGCCAC TGGTAACAGG ATTAGCAGAG CGAGGTATGT 14820 AGGCGGTGCT ACAGAGTTCTTGAAGTGGTG GCCTAACTAC GGCTACACTA GAAGGACAGT 14880 ATTTGGTATC TGCGCTCTGCTGAAGCCAGT TACCTTCGGA AAAAGAGTTG GTAGCTCTTG 14940 ATCCGGCAAA CAAACCACCGCTGGTAGCGG TGGTTTTTTT GTTTGCAAGC AGCAGATTAC 15000 GCGCAGAAAA AAAGGATCTCAAGAAGATCC TTTGATCTTT TCTACGGGGT CTGACGCTCA 15060 GTGGAACGAA AACTCACGTTAAGGGATTTT GGTCATGAGA TTATCAAAAA GGATCTTCAC 15120 CTAGATCCTT TTAAATTAAAAATGAAGTTT TAAATCAATC TAAAGTATAT ATGAGTAAAC 15180 TTGGTCTGAC AGTTACCAATGCTTAATCAG TGAGGCACCT ATCTCAGCGA TCTGTCTATT 15240 TCGTTCATCC ATAGTTGCCTGACTCCCCGT CGTGTAGATA ACTACGATAC GGGAGGGCTT 15300 ACCATCTGGC CCCAGTGCTGCAATGATACC GCGAGACCCA CGCTCACCGG CTCCAGATTT 15360 ATCAGCAATA AACCAGCCAGCCGGAAGGGC CGAGCGCAGA AGTGGTCCTG CAACTTTATC 15420 CGCCTCCATC CAGTCTATTAATTGTTGCCG GGAAGCTAGA GTAAGTAGTT CGCCAGTTAA 15480 TAGTTTGCGC AACGTTGTTGCCATTGCTAC AGGCATCGTG GTGTCACGCT CGTCGTTTGG 15540 TATGGCTTCA TTCAGCTCCGGTTCCCAACG ATCAAGGCGA GTTACATGAT CCCCCATGTT 15600 GTGCAAAAAA GCGGTTAGCTCCTTCGGTCC TCCGATCGTT GTCAGAAGTA AGTTGGCCGC 15660 AGTGTTATCA CTCATGGTTATGGCAGCACT GCATAATTCT CTTACTGTCA TGCCATCCGT 15720 AAGATGCTTT TCTGTGACTGGTGAGTACTC AACCAAGTCA TTCTGAGAAT AGTGTATGCG 15780 GCGACCGAGT TGCTCTTGCCCGGCGTCAAT ACGGGATAAT ACCGCGCCAC ATAGCAGACC 15840 TTTAAAAGTG CTCATCATTGGAAAACGTTC TTCGGGGCGA AAACTCTCAA GGATCTTACC 15900 GCTGTTGAGA TCCAGTTCGATGTAACCCAC TCGTGCACCC AACTGATCTT CAGCATCTTT 15960 TACTTTCACC AGCGTTTCTGGGTGAGCAAA AACAGGAAGG CAAAATGCCG CAAAAAAGGG 16020 AATAAGGGCG ACACGGAAATGTTGAATACT CATACTCTTC CTTTTTCAAT ATTATTGAAG 16080 CATTTATCAG GGTTATTGTCTCATGAGCGG ATACATATTT GAATGTATTT AGAAAAATAA 16140 ACAAATAGGG GTTCCGCGCACATTTCCCCG AAAAGTGCCA CCTGACGTCG ACGGATCGGG 16200 AGATCTAATG AAAGACCCCACCTGTAGGTT TGGCAAGCTA GCTTAAGTAA CGCCATTTTG 16260 CAAGGCATGG AAAAATACATAACTGAGAAT AGAGAAGTTC AGATCAAGGT CAGGAACAGA 16320 TGGAACAGCT GAATATGGGCCAAACAGGAT ATCTGTGGTA AGCAGTTCCT GCCCCGGCTC 16380 AGGGCCAAGA ACAGATGGAACAGCTGAATA TGGGCCAAAC AGGATATCTG TGGTAAGCAG 16440 TTCCTGCCCC GGCTCAGGGCCAAGAACAGA TGGTCCCCAG ATGCGGTCCA GCCCTCAGCA 16500 GTTTCTAGAG AACCATCAGATGTTTCCAGG GTGCCCCAAG GACCTGAAAT GACCCTGTGC 16560 CTTATTTGAA CTAACCAATCAGTTCGCTTC TCGCTTCTGT TCGCGCGCTT CTGCTCCCCG 16620 AGCTCAATAA AAGAGCCCACAACCCCTCAC TCGGGG 16656 24 base pairs nucleic acid single linear 2ATCTCTACGG TGGTCCTAAA TAGT 24 42 base pairs nucleic acid single linear 3TATATTCTAG ATTTTTTTTT TTTTTTTTTT TTTTTTGAAA TG 42 48 base pairs nucleicacid single linear 4 TATATGGGCC CGATTTAGGT GACACTATAG ATTGACGGCGTAGTACAC 48 23 base pairs nucleic acid single linear 5 CTGGCAACCGGTAAGTACGA TAC 23 21 base pairs nucleic acid single linear 6 ATACTAGCCACGGCCGGTAT C 21 21 base pairs nucleic acid single linear 7 TCCTCTTTCGACGTGTCGAG C 21 21 base pairs nucleic acid single linear 8 ACCTTGGAGCGCAATGTCCT G 21 21 base pairs nucleic acid single linear 9 CCTTTTCAGGGGATCCGCCA C 21 21 base pairs nucleic acid single linear 10 GTGGCGGATCCCCTGAAAAG G 21 20 base pairs nucleic acid single linear 11 TGGGCCGTGTGGTCGTCATG 20 21 base pairs nucleic acid single linear 12 TGGGTCTTCAACTCACCGGA C 21 22 base pairs nucleic acid single linear 13 CAATTCGACGTACGCCTCAC TC 22 22 base pairs nucleic acid single linear 14 GAGTGAGGCGTACGTCGAAT TG 22 33 base pairs nucleic acid single linear 15 TATATAGATCTAATGAAAGA CCCCACCTGT AGG 33 40 base pairs nucleic acid single linear 16TCAATCCCCG AGTGAGGGGT TGTGGGCTCT TTTATTGAGC 40 36 base pairs nucleicacid single linear 17 CCACAACCCC TCACTCGGGG ATTGACGGCG TAGTAC 36 23 basepairs nucleic acid single linear 18 CTGGCAACCG GTAAGTACGA TAC 23 22 basepairs nucleic acid single linear 19 GGTAACAAGA TCTCGTGCCG TG 22 53 basepairs nucleic acid single linear 20 TATATATATA TGCGGCCGCT TTCTTTTATTAATCAACAAA ATTTTGTTTT TAA 53 48 base pairs nucleic acid single linear 21TATATGAGCT CTTTTTTTTT TTTTTTTTTT TTTTTTGAAA TGTTAAAA 48 34 base pairsnucleic acid single linear 22 TATATCTCGA GGGTGGTGTT GTAGTATTAG TCAG 3443 base pairs nucleic acid single linear 23 TATATGGGCC CTTAAGACCATCGGAGCGAT GCTTTATTTC CCC 43 18 base pairs nucleic acid single linear 24TCTCTACGGT GGTCCTAA 18 5 amino acids amino acid single linear 25 Ser LeuArg Trp Ser 1 5 26 base pairs nucleic acid single linear 26 CATCTCTACGGTGGTCCTAA ATAGTC 26 34 base pairs nucleic acid single linear 27TCGAGACTAT TTAGGACCAC CGTAGAGATG GGCC 34 25 base pairs nucleic acidsingle linear 28 CCCTTGTACG GCTAACCTAA AGGAC 25 33 base pairs nucleicacid single linear 29 TCGAGTCCTT TAGGTTAGCC GTACAAGGGG GCC 33 26 basepairs nucleic acid single linear 30 CATCGCTACG GTGGTCCTAA ATAGTC 26 34base pairs nucleic acid single linear 31 TCGAGACTAT TTAGGACCACCGTAGCGATG GGCC 34 48 base pairs nucleic acid single linear 32CGGAAATAAA GCATCTCTAC GGTGGTCCTA AATAGTCAGC ATAGTACC 48 56 base pairsnucleic acid single linear 33 TCGAGGTACT ATGCTGACTA TTTAGGACCACCGTAGAGAT GCTTTATTTC CGGGCC 56 41 base pairs nucleic acid single linear34 TATATGCGGC CGCTCTAGAT TACAATTTGG ACTTTCCGCC C 41 44 base pairsnucleic acid single linear 35 TATATATGAG CTCTTACAAA TAAAGCAATAGCATCACAAA TTTC 44 36 base pairs nucleic acid single linear 36TATATGAATT CGTTTGGACA AACCACAACT AGAATG 36 44 base pairs nucleic acidsingle linear 37 TATATATGAG CTCTAATAAA ATGAGGAAAT TGCATCGCAT TGTC 44 43base pairs nucleic acid single linear 38 TATATGAATT CATAGAATGACACCTACTCA GACAATGCGA TGC 43 46 base pairs nucleic acid single linear 39TATATGAGCT CGGGTCGGCA TGGCATCTCC ACCTCCTCGC GGTCCG 46 52 base pairsnucleic acid single linear 40 TCCACCTCCT CGCGGTCCGA CCTGGGCATCCGAAGGAGGA CGCACGTCCA CT 52 48 base pairs nucleic acid single linear 41TATATGAGCT CCTCCCTTAG CCATCCGAGT GGACGTGCGT CCTCCTTC 48 47 base pairsnucleic acid single linear 42 TATATGCGGC CGCTTTCTTT TATTAATCAACAAAATTTTG TTTTTAA 47 37 base pairs nucleic acid single linear 43TATATGAGCT CGAAATGTTA AAAACAAAAT TTTGTTG 37 34 base pairs nucleic acidsingle linear 44 TATATATAGA TCTTTGACAT TGATTATTGA CTAG 34 42 base pairsnucleic acid single linear 45 CCGTCAATAC GGTTCACTAA ACGAGCTCTGCTTATATAGA CC 42 38 base pairs nucleic acid single linear 46 GCTCGTTTAGTGAACCGTAT TGACGGCGTA GTACACAC 38 33 base pairs nucleic acid singlelinear 47 TATATATAGA TCTGGTGTGG AAAGTCCCCA GGC 33 31 base pairs nucleicacid single linear 48 CTACGCCGTC AATGCCGAGG CGGCCTCGGC C 31 37 basepairs nucleic acid single linear 49 GGCCGCCTCG GCATTGACGG CGTAGTACACACTATTG 37 41 base pairs nucleic acid single linear 50 TATATATCTCGAGAAGCTCT AAGGTAAATA TAAAATTTAC C 41 38 base pairs nucleic acid singlelinear 51 TATATATCTC GAGAGGTTGG AATCTAAAAT ACACAAAC 38 43 base pairsnucleic acid single linear 52 TATATATGCG GCCGCAAGCT CTAAGGTAAATATAAAATTT ACC 43 40 base pairs nucleic acid single linear 53 TATATATGCGGCCGCAGGTT GGAATCTAAA ATACACAAAC 40 35 base pairs nucleic acid singlelinear 54 TCGAGCACGT GGCGCGCCTG ATCACGCGTA GGCCT 35 35 base pairsnucleic acid single linear 55 CTAGAGGCCT ACGCGTGATC AGGCGCGCCA CGTGC 3535 base pairs nucleic acid single linear 56 TATATCTCCA GATGAGGTACATGATTTTAG GCTTG 35 40 base pairs nucleic acid single linear 57TATATATCGA TTCAAGGCAT TTTCTTTTCA TCAATAAAAC 40 35 base pairs nucleicacid single linear 58 TATATCTCCA GATGATGACA ATGTGGTGTC TGACG 35 32 basepairs nucleic acid single linear 59 TATATATCGA TTCATGACGA CCGGACCTTG CG32 28 base pairs nucleic acid single linear 60 TATATGGGCC CCCCCCCCCCCCCCAACG 28 30 base pairs nucleic acid single linear 61 TATATATCGATCCCCCCCCC CCCCCCAACG 30 34 base pairs nucleic acid single linear 62TATATCCATG GCTTACAATC GTGGTTTTCA AAGG 34 33 base pairs nucleic acidsingle linear 63 TATATGGGCC CTCGATGAGT CTGGACGTTC CTC 33 33 base pairsnucleic acid single linear 64 TATATATCGA TTCGATGAGT CTGGACGTTC CTC 33 37base pairs nucleic acid single linear 65 TATATCCATG GATCCAATTTGCTTTATGAT AACAATC 37 30 base pairs nucleic acid single linear 66TATATGGGCC CGGTCGACGC CGGCCAAGAC 30 30 base pairs nucleic acid singlelinear 67 TATATATCGA TGGTCGACGC CGGCCAAGAC 30 32 base pairs nucleic acidsingle linear 68 TATATCCATG GTGCCAGCCA GTTGGGCAGC AG 32 23 base pairsnucleic acid single linear 69 TTAATTAACG GCCGCCACCA TGG 23 13 base pairsnucleic acid single linear 70 TAACGGCCGC CAC 13 20 base pairs nucleicacid single linear 71 CCATGGTGGC GGCCGTTAAT 20 16 base pairs nucleicacid single linear 72 GGTTTAAACA GGAGCT 16 16 base pairs nucleic acidsingle linear 73 CCTGTTTAAA CCAGCT 16 47 base pairs nucleic acid singlelinear 74 TATATGCGGC CGCACCACCA CCATGAATAG AGGATTCTTT AACATGC 47 34 basepairs nucleic acid single linear 75 TATATGCGGC CGCTCATCTT CGTGTGCTAGTCAG 34 61 base pairs nucleic acid single linear 76 TATATGCGGCCGCATCTCTA CGGTGGTCCT AAATAGTACC ACCACCATGA ATAGAGGATT 60 C 61 25 basepairs nucleic acid single linear 77 CTCATCGATC AGATCTGACT AGTTG 25 33base pairs nucleic acid single linear 78 GATCCAACTA GTCAGATCTGATCGATGAGG GCC 33 56 base pairs nucleic acid single linear 79 ACTTATCGATGGTTCTAGAC TCCCTTAGCC ATCCGAGTGG ACGTGCGTCC TCCTTC 56 52 base pairsnucleic acid single linear 80 TCCACCTCCT CGCGGTCCGA CCTGGGCATCCGAAGGAGGA CGCACGTCCA CT 52 57 base pairs nucleic acid single linear 81TCGGACCGCG AGGAGGTGGA GATGCCATGC CGACCCATTG ACGGCGTAGT ACACACT 57 36base pairs nucleic acid single linear 82 CTGGACTAGT TAATACTGGTGCTCGGAAAA CATTCT 36 40 base pairs nucleic acid single linear 83GTCAAGCTTG CTAGCTACAA CACCACCACC ATGAATAGAG 40 40 base pairs nucleicacid single linear 84 CAGTCTCGAG TTACTACCAC TCTTCTGTCC CTTCCGGGGT 40 43base pairs nucleic acid single linear 85 TATATGCGGC CGCACCACCATGTCCGCAGC ACCACTGGTC ACG 43 34 base pairs nucleic acid single linear 86TATATAGATC TCTTGATCAG CTTCAGAAGA TGGC 34 24 base pairs nucleic acidsingle linear 87 TCAATGGCGG GAAGAGGCGG TTGG 24 31 base pairs nucleicacid single linear 88 CCGCCTCTTC CCGCCATTGA CGGCGTAGTA C 31 34 basepairs nucleic acid single linear 89 TATATAGATC TCTTGATCAG CTTCAGAAGATGGC 34 44 base pairs nucleic acid single linear 90 TATATATATGCGGCCGCACC GCCAAGATGT TCCCGTTCCA GCCA 44 38 base pairs nucleic acidsingle linear 91 TATATATATG CGGCCGCTCA ATTATGTTTC TGGTTGGT 38 35 basepairs nucleic acid single linear 92 CTCGAGCTCG AGGCACCAGC ACCATGCAACTTTTT 35 29 base pairs nucleic acid single linear 93 CTACTAGATCCCTAGATGCT GGATCTTCC 29 29 base pairs nucleic acid single linear 94GGAAGATCCA GCATCTAGGG ATCTAGTAG 29 26 base pairs nucleic acid singlelinear 95 GGGCGATATC AAGCTTATCG ATACCG 26 26 base pairs nucleic acidsingle linear 96 GGGCGATATC AAGCTTATCG ATACCG 26 19 base pairs nucleicacid single linear 97 AATACGACTC ACTATAGGG 19 29 base pairs nucleic acidsingle linear 98 CTACTAGATC CCTAGATGCT GGATCTTCC 29 17 base pairsnucleic acid single linear 99 ATTAACCCTC ACTAAAG 17 29 base pairsnucleic acid single linear 100 GGAAGATCCA GCATCTAGGG ATCTAGTAG 29 17base pairs nucleic acid single linear 101 ATTAACCCTC ACTAAAG 17 19 basepairs nucleic acid single linear 102 AATACGACTC ACTATAGGG 19 34 basepairs nucleic acid single linear 103 CCTCGAGCTC GAGCTTGGGT GGCTTTGGGGCATG 34 17 base pairs nucleic acid single linear 104 ATTACCCCTC ACTAAAG17 44 base pairs nucleic acid single linear 105 CCCTCGAGCT CGAGGGGTCACTGAGAAACT AGAAAAAGAA TTAG 44 37 base pairs nucleic acid single linear106 CCGCGGCCGC GTATCTGTGG GAGCCTCAAG GGAGAAC 37 44 base pairs nucleicacid single linear 107 CGCGCGGGCC CTGTGACATT GAATAGAGTG AGGGTCCTGT TGGG44 45 base pairs nucleic acid single linear 108 AAAGGTTTCA CATTTGTAGCTTGCTGTGTC ATTGCGATCT CTACG 45 45 base pairs nucleic acid single linear109 GTGGTCCTAA ATAGTTCACT CTATTCAATG TCACACTCGA GCCGG 45 33 base pairsnucleic acid single linear 110 TATATTCTAG AGCAAGCAAC AGTTACTGCG ACG 3333 base pairs nucleic acid single linear 111 TATATATCGA TCCGAAGCGTAGAGTCACAC TTG 33 18 base pairs nucleic acid single linear 112TTAACTGTCA AAAGCCAC 18 68 base pairs nucleic acid single linear 113CGATGTGGCT TTTAGATGTT AAACCAGAGA AACACACGGA CTTCGGTCCG TGGTATATTA 60GCTGGTAT 68 70 base pairs nucleic acid single linear 114 CTAGATACCAGCTAATATAC CACGGACCGA AGTCCGTGTG TTTCTCTGGT TTAACATCTA 60 AAAGCCACAT 7042 base pairs nucleic acid single linear 115 TATATCTCGA GACCACCATGAGTGCTGTAA GTAATAGGAA GC 42 36 base pairs nucleic acid single linear 116TATATCTCGA GCTAGAAGGC AAACCTAACA CCCAAC 36 31 base pairs nucleic acidsingle linear 117 TATATGGGCC CTACATGTCC CACTGTTCAA G 31 31 base pairsnucleic acid single linear 118 TATATGGGCC CGTACGGAAG GAAAGAAGTC A 31 32base pairs nucleic acid single linear 119 TATATGGGCC CATTTTGGTTTTGCTATGCG TA 32 16 base pairs nucleic acid single linear 120 TCTCTGTCCTCCATGA 16 66 base pairs nucleic acid single linear 121 TCGAGTCATGGAGAGAGGAG AACCAGAGAA ACACACGGAC TTCGGTCCGT GGTATATTAC 60 CTGGAT 66 64base pairs nucleic acid single linear 122 CGATCCAGGT AATATACCACGGACCGAAGT CCGTGTGTTT CTCTGGTTCT CCTCTCTCCA 60 TGAC 64 35 base pairsnucleic acid single linear 123 GCCTCGAGAC AATGTACAGG ATGCAACTCC TGTCT 3536 base pairs nucleic acid single linear 124 GAATCGATTT ATCAAGTCAGTGTTGGAGAT GATGCT 36 31 base pairs nucleic acid single linear 125TATATGGGCC CATCGAGGTG AGAAAGAGGA C 31 31 base pairs nucleic acid singlelinear 126 TATATGGGCC CTGTATCTGG CGGACCCGTG G 31 31 base pairs nucleicacid single linear 127 TATATGGGCC CGCAGACAAG ACGCGCGGCG C 31 24 basepairs nucleic acid single linear 128 AUCUCUACGG UGGUCCUAAA UAGU 24

We claim:
 1. A method for producing one or more recombinant proteins,comprising growing, under suitable nutrient conditions, an isolated,cultured eukaryotic host cell transformed or transfected with aeukaryotic layered vector initiation system in a manner allowingexpression of the recombinant protein, wherein said eukaryotic layeredvector initiation system comprises a eukaryotic promoter 5′ of viralcDNA which initiates within said cell the 5′ to 3′ synthesis of RNA fromsaid cDNA, wherein said RNA comprises a vector which autonomouslyamplifies in said cell and expresses a heterologous nucleic acidsequence which encodes said recombinant protein, and wherein said vectorwhich autonomously amplifies in a cell comprises a sequence whichinitiates transcription of alphavirus RNA, a nucleic acid sequence whichencodes alphavirus nonstructural proteins, and an alphavirus RNApolymerase recognition sequence.
 2. A method for delivering aheterologous nucleic acid sequence to an isolated, cultured eukaryotichost cell, comprising administering to said isolated, culturedeukaryotic host cell a eukaryotic layered vector initiation system,wherein said eukaryotic layered vector initiation system comprises aeukaryotic promoter 5′ of viral cDNA which initiates within said cellthe 5′ to 3′ synthesis of RNA from said cDNA, wherein said RNA comprisesa vector which autonomously amplifies in said cell and expresses aheterologous nucleic acid sequence which encodes said recombinantprotein, and wherein said vector which autonomously amplifies in a cellcomprises a sequence which initiates transcription of alphavirus RNA, anucleic acid sequence which encodes alphavirus nonstructural proteins,and an alphavirus RNA polymerase recognition sequence.
 3. The methodaccording to claim 1 or 2 wherein said vector which autonomouslyamplifies in said cell further comprises a polyadenylate tract.
 4. Themethod according to claim 1 or 2 wherein said eukaryotic promoter is anRNA polymerase II promoter.
 5. The method according to claim 1 or 2wherein said vector which autonomously amplifies in said cell furthercomprises an alphavirus subgenomic promoter.
 6. The method according toclaim 1 or 2 wherein said eukaryotic promoter is selected from the groupconsisting of the MoMLV promoter, metallothionein promoter,glucocorticoid promoter, SV40 promoter, and CMV promoter.
 7. The methodaccording to claim 1 or 2 wherein said heterologous nucleic acidsequence encodes a lymphokine.
 8. The method according to claim 7wherein said lymphokine is selected from the group consisting of IL-1,IL-2, IL-3, IL-4, Il-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12,IL-13, IL-14, IL-15, .alpha.-IFN, .beta.-IFN, .gamma.-IFN G-CSF, andGM-CSF.
 9. The method according to claim 1 or 2 wherein saidheterologous sequence is selected from the group consisting of: anantigen from a virus, an antigen from a bacteria, an antigen from afungus and an antigen from a parasite.
 10. The method according to claim9 wherein said viral antigen is obtained from a virus selected from thegroup consisting of influenza virus, respiratory syncytial virus, HPV,HBV, HCV, EBV, HIV, HSV, Fel, V, FIV, Hantavirus, HTLV I, HTLV II, andCMV.
 11. The method according to claim 1 or 2 wherein said heterologoussequence is a non-tumorigenic antigen associated with tumorigenic cells.12. The method according to claim 1 or 2 wherein said eukaryotic layeredvector initiation system further comprises a transcription terminationsequence.
 13. The method according to claim 1 or 2 wherein said cDNA ofsaid eukaryotic layered vector initiation system further comprises aribozyme sequence.
 14. The method according to claim 13 wherein saidribozyme sequence is a hepatitis delta virus ribozyme sequence.